Molecular Characterization And Biological Functional Activities Of Interferon Gamma In Large Yellow Croaker(Larimichthys Crocea)

Interferon gamma（IFN-γ）is a Th1 cytokine that plays a very important role in almost all phases of immune and inflammatory responses.Studies have shown that IFN-γ is a kind of pleiotropic cytokine,and can increase anti-viral,anti-tumour defences,enhance antigen presentation,activate and mediate the inflammatory response,promote the Th1 immune response and regulate cell proliferation and apoptosis.Recently,a variety of IFN-γ have been successfully cloned from fish,while another subtype of IFN-γ called IFN-γ rel had been found in some fish.For fish the research on IFN-γ mainly focused on its molecular characteristics and expression.Relative to the mammals,the current understanding of fishIFN-γfunction is not enough,it is necessary to conduct a comprehensive and in-depth study on different fishIFN-γ.In this paper,we have done some research on the characteristics and functions on the large yellow croakerIFN-γ（ LycIFN-γ）,We hope to provide theoretical and technical supports for the disease prevention and treatment in the large yellow croaker.In this study,we cloned LycIFN-γ gene.The full-length of LycIFN-γ sequence was 1466 bp.The open reading frame encoded a 200 aa peptide.The mature protein contained a signature motif V¹⁵⁷QSKALWELSGI¹⁶⁸ and a nuclear localization signal（NLS）motif consisits of four basic amino acids at the C-terminal end of the protein（RRRR）.Tissue expression analysis showed that LycIFN-γ was constitutively expressed in brain,intestine,head kidney,skin,gills,liver,spleen,heart and blood tested. LycIFN-γ was also detected in several immune related cells such as primary neutrophil-like cells,macrophages,head kidney leukocytes and large yellow croaker head kidney cell line（LYCK）.Based on stimulation with polyinosinic-polycytidylic acid[poly（I:C）]or inactivated trivalent bacterial vaccine, LycIFN-γwas significantly increased in spleen and head kidney. LycIFN-γwas significantly upregulated by poly（I:C）or lipopolysaccharide（LPS）in head kidney leukocytes.Recombinant LycIFN-γ protein （rLycIFN-γ） produced in Escherichia coli could enhance respiratory burst responses in macrophages.Furthermore rLycIFN-γ could not only upregulate the transcriptional level of iNOS and release of nitric oxide（NO）,but also upregulated the transcriptional levels of proinflammatory cytokines TNF-α and IL-1βin macrophages,indicating that rLycIFN-γhas a role in mediating inflammatory response.In addition,rLycIFN-γcould significantly upregulate the transcriptional levels of IFN-γreceptor CRFB13,signal transduction factor STAT1,transcription factors IRF1 and T-bet and Th1 related cytokines IFN-γand IL-2 in head kidney leukocytes,suggesting that LycIFN-γmay have the potential to promote Th1 immune response in large yellow croaker.Taken together,our results show that LycIFN-γmay have functional activities similar to its mammalian counterpart.