Expression of biologically active bovine interleukin 7 and evaluating the activity in vitro

Abstract

Aim: Interleukin 7 (IL-7) is a Υc family cytokine involved in the homeostatic proliferation and maintenance of immune cells. In the present study, we report the expression of bovine IL-7 (bIL-7) in Escherichia coli and evaluated for its biological activity. Materials and Methods: The sequence coding for bIL-7 (mature protein) was amplified from primary bovine kidney cell culture and cloned into pET28-a vector and expressed in E.coli (BL 21 DE3). The expressed protein was purified by nickelnitrilotriacetatechromatography, and the reactivity of the protein was confirmed by western blotting using monoclonal antibodies raised against human IL-7. The biological activity of expressed bIL-7 was evaluated by analyzing its effect on the expression of anuclear factor for activated T-cells c1 (NFATc1), B-cell lymphoma 2 (Bcl2), suppressor of cytokine signaling 3 (SOCS3) molecules in bovine peripheral blood mononuclear cells (PBMCs) by quantitative polymerase chain reaction. Ability of the expressed protein was also analyzed by its effect on phosphorylating signal transducer and activator 3 (STAT3) molecule by immunostaining in human embryonic kidney cells 293 (HEK293) cells. Results: The bIL-7 was able to induce the expression of Bcl2 and NFATc1expression in bovine PBMCs by 7 and 5-folds, respectively, whereas a 2-fold decrease was observed in the case of SOCS3 expression. Immunostaining studies in HEK293 cells using antihuman phospho-STAT3 showed activation and nuclear translocation of STAT3 molecule on bIL-7 treatment. Conclusion: bIL-7 gene was successfully amplified, cloned, and expressed in a prokaryotic expression system. The biological activity study showed that the E.coli expressed bIL-7 protein is biologically active. Considering the role of IL-7 in T-cell homeostasis and memory cell generation, this molecule can be used for enhancing the vaccine response and that has to be proved by further experiments.