Cloning, Characterization And Expression Analysis Of Interferon γ-2β Genomic DNA From Common Carp(Cyprinus Carpio L.)

Cytokines plays a vital role in regulation reaction of the immune system,it wasfound in the study of the immune system of higher animals. Interferon gamma (IFN-γ)is a pleiotropic proinflammatory and antiviral cytokine that is produced primarily byactivated Th1T helper cells[1], cytotoxic T lymphocytes of the TC1phenotype[2]andnatural killer (NK) cells[3],it is only active as a homodimer[4-6]. This cytokine was firstidentified in PHA-activated mammalian lymphocyte supernatants and shown to haveunique antiviral properties[7]. In addition to orchestrating antiviral responses[8-9],IFN-γregulates the production of an array of cytokines that promote a Th1phenotype[1]andskewing of T cells toward a Th1modality[10-11]. Interferon gamma is a centralparticipant in host resistance to various infections[12-14], demonstrated using IFN-γknockout mice infected with Leishmania major[15], Listeria monocytogenes[16]andMycobacteria[17]. In this study, the IFNγ-2β encoding sequence and its genomicstructure are acquired, and the transcription differences after stimulation in varioustimes are also analyzed. The research laid the foundation for further studying theexpression manner、function characteristic and regulation mechanism of IFNγ-2β invivo and the action mechanism in the inflammatory reaction,emergency reaction andimmune response.The study used part DNA sequence obtained from EST sequencing and labeledwith DIG used as a probe to filter common carp IFNγ-2β from peripheral bloodleukocyte cDNA library that was stimulated by mitogen. Then we got the full-lengthcDNA sequence of carp IFNγ-2β.The GenBank number: JX181980, the sequencecontains a119bp5’-UTR and a218bp3’-UTR, the open reading frame (ORF) of thisgene was537bp which putatively encodes178amino acids length of873bp andthere are several motifs for mRNA instability (ATTTA) in the3’-untranslated. Wehave designed two prime numbers of IFNγ-2β cDNA obtained and the use of non- coding region of the spleen genomic DNA to the PCR as a board. These twosequences are common, and the genomic sequence of the IFNγ-2β. It is a length of2084bp, including four exons and three introns, which is in accordance with the IFNγ-2β and intron splice sites are in line with the supervision of the GT-AG. We stimulatedcarp peripheral blood leukocytes using the mitosis of the original (ConA、PHA andLPS) and abstract total RNA of different types of stimulation at different times, thenprimers were designed on the basis of carp IFNγ-2β full-length cDNA and β-actinsequence, then, the IFNγ-2β of the expression of these groups was analyzed. Theresults show that: the carp IFNγ-2β transcription level increased in mitogen-stimulated leukocyte through the RT-PCR. Expecially the raising expression level ofjoint stimulation by PHA and LPS was more obvious and the enhancement extentgradually rised over time. It not only suggests that IFNγ-2β transcription has differentsensitivities to different inducers, but also explains that IFNγ-2β play a immune rolein cells stimulated early.