Molecular Cloning Of RORγ And STAT3 And Immune Function Effect On Th17 Cells In Common Carp (Cyprinus Carpio L.)

The helper T cell is a kind of T cells which can constitutive express the TCRαβ and CD4 molecule. According to the different kind of cytokines secreted by CD4+ T cells in the case of stimulated by pathogens.The helper T cells are divided into type helper T cells(Th1), typeⅠ Ⅱ(Th2) helper T cells and Th17 cells. Th17 cells can mainly produce IL-17(IL-17 A and IL-17F), which are members of natural immune group in adaptive immunity and natural immunity against inflammatory response in the construction of the first line of defense work, and its mostly functions are made by its secretion, namely, IL-17.Th 17 cells play roles in recruiting of neutrophils, activation of innate immune cells, strengthening the function of B cells, promoting inflammatory cytokines release such as TNF and IL-1β.In addition, the inflammation cytokines and other factors such as intercellular adhesion molecules, antimicrobial peptides and the metalloproteinase can be induced to secrete through the IL-17 signaling pathway.Th17 cells are induced to differentiate into mature Th17 cells by IL-6,TGF-β, IL- 21 and IL – 23.This process also depends on the activation of STAT3(signaling and transcriptional activation factor) and RORα and RORγ(retinoic acid related orphan receptor), the transcriptional activity of RORγ are regulated by STAT3 at the same time.In bony fish, there are only ROR genes in zebra fish and in grass fish have been identified, including RORα1, RORα2, RORβ, RORγ1 and RORγ2. Researchers have already studied these genes expression in the development process of zebra fish, but there is little research of the immunology function of bony fish in immune regulation. Although there are relatively more studies of STAT3 in bony fish, the gene cloning of STAT3 is not reported in common carp.The research model in this paper is common carp. First of all, we designed the primers according to the conservative district of STAT3 and RORγ for cloning pieces. We have got a total length of 2157 bp complete ORF(open reading frame) of STAT3 and RORγ genetic code fragment of 732 bp in common carp. With the help of SMART software protein prediction analysis, we determined the STAT3 protein contains a STAT-int(2-122) structural domain and a characteristic SH2 domain(583-664) and two spiral structure domain(136-223,268-292). The RORγ gene contains 732 bp nucleotides which encode 243 amino acids. There is a structural domain named HOLI(46-202) in the sequence. We built system evolutionary trees on RORγ and STAT3 and found that carp has the most similar relationships with the grass carp in RORγ; they locate in the same branch of the evolutionary tree. But the genetic relationship of STAT3 is closer to zebra fish.For further studying the immune function on the cell cytokines secreted by Th17 cells, we selected the IL-17 AF2 cytokine genes for this research. We detected the organization distribution through the fluorescence quantitative PCR analysis, and the results showed that the highest IL-17 AF2 expression located in the head kidney, follow by the liver, spleen, skin, foregut, hindgut, and gill. For studying the expression changes of IL-17AF2 after virus infection, we injected PolyI: C into stimulation carp, and detected the mRNA level at different time after injection. The results showed the IL-17 AF2 gene expression had been raised commonly in carp.The level was significantly up-regulated in head kidney and skin, while the elevation in the gills was small. Another test results showed in the IL17-AF2 gene expression to raise the most obvious group and period, the content of STAT3 and RORγ have increased accordingly. Based on test results shows that IL- 17 af2 associated with the antiviral immune response of carp, and STAT3 and RORγ could be associated with the function of Th17 cells.