Biotechnology

Biography

Biography: Sheetal Saini

Abstract

Research focusing on evaluating equine cytokines relied on the detection of mRNA rather than proteins due to limited availability of equine cytokines. Thus, the present work was undertaken to obtain biologically active recombinant equine IL-18 and IFN-γ. The coding sequences of equine IL-18 and IFN-γ gene were cloned and identity was confirmed by sequence analysis. Subsequently, recombinant equine IL-18 and IFN-γ were expressed in Escherichia coli and proteins were purified by Nickel-NTA chromatography. Biological activity of recombinant cytokines was assessed by lymphocyte proliferation assay, cytokine ELISA and real time RT-PCR. For lymphocyte proliferation assay, 4×105 equine peripheral blood mononuclear cells (PBMCs) were cultured per well in 96 well culture plate and stimulated with different concentration of recombinant cytokines and Concanavalin A (5 µg/ml) as positive control. At 72 hours post stimulation, cell proliferation assay was performed by XTT method. Recombinant equine IL-18 and IFN-γ induced significant proliferation at 500 ng/ml concentration. Secretion of IL-10 and IFN-γ by activated PBMCs was assessed by ELISA at protein level and by real-time PCR at mRNA level. Recombinant IL-18 was able to induce the production of both IL-10 and IFN-γ (100 pg/ml) in equine PBMC while IFN-γ induced IL-10 secretion (100 pg/ml). On the other hand, IL-18 induced IFN-γ & IL-10 transcripts by 128 fold and IL-2 & IL-4 by 64 fold while IFN-γ induced transcription of IL-10 by 128 fold, IL-4 by 64 fold and IL-2 by 32 fold. Results indicate that recombinant equine cytokines activate the immune cells and stimulate the secretion of natural cytokines.