Recombinant Expression, Purification And Immunological Function Of Grass Carp IFN-γrel

CD4+T lymphocytes can be divided into four cell subsets (Th1, Th2, Th17andTreg）according to specific transcription factors and released cytokines. Transcriptionfactor T-bet is specific for Th1cell subsets which mainly secrete IFN-and IL-12.IFN-producted by DC cells can up-regulate the transcriptional level of T-bet and thenpromote CD4+T cell differentiation into Th1cell subsets. Moreover, T-bet canstimulate Th1cell to secrete IFN-by binding to the promoter of IFN-gene. Howeverthere are two type II interferon genes in teleost, IFN-γ2and IFN-γrel (can also becalled IFN-γ1), and the immune function of the latter is unclear now. To clarify thefunctional role of fish IFN-γrel, IFN-γrel cDNA was isolated from grass carp and thesequence encoding the putative mature IFN-γrel was cloned into the bacterialexpression vector pET30a(+). Results showed that recombinant grass carp IFN-γrelwas primarily produced in the form of inclusion body and then the soluble recombinantproteins were obtained by optimizing conditions of denaturation and renaturation.However, the purified protein by gel chromatography and affinity chromatography wasnot shown biological activity. Subsequently, recombinant grass carp IFN-γrel wasproduced in eukaryotic expression system by using pcDNA3.1-myc-his vector inHEK293cells and the over-expressed protein IFN-γrel was shown to significantlyup-regulate IL-8mRNA expression in grass carp head kidney leukocytes, confirmingthe biological activity of recombinant proteins. Regarding the close relationship ofT-bet and IFN-γ in Th1cells, grass carp T-bet was cloned and identified. Multipleamino acid sequence alignment of grass carp T-bet with the homologous proteins fromsome representative species and phylogenetic tree analysis showed that what we havegot was a real grass carp T-bet gene. Meanwhile, the polyclonal antibody was raised inthe rabbit against grass carp T-bet and its specificity was determined by using WBanalysis, providing supports for determining its protein levels in the followingexperiments. In summary, recombinant expression, protein purification and functionalinvestigation of grass carp IFN-γrel have been carried out. And also, grass carp T-betwas isolated and identified. These results not only reveal evolutional information of structure and function of fish IFN-γ, but also provide basis for evaluating the existenceof fish Th1cells.