ABSTRACT
Objective To construct a eukaryotic expression vector pEGFP N1/IL-37b of full-length and mature IL-37b,and to detect the expression of both full-length and mature IL-37b in RAW 264.7 cells, a mouse macrophage cell line. Methods To construct the eukaryotic vectors of full-length and mature IL-37b by using plasmid pUBC/IL-37b as a template containing the coding region of IL-37b full-length gene. To detect the expression of IL-37b by western blot and confocal microscopy after transfected the recombinant plasmid into RAW 264.7 cells, and to detect the inhibition of full-length and mature IL-37b on IL-6 production by real-time PCR. Results Eukaryotic vectors pEGFP N1/IL-37b expressed full-length and mature IL-37b after transfection in cells, which inhibited LPS-induced IL-6 production. Conclusions Eukaryotic vectors of full-length and mature IL-37b can be successfully constructed,and lays a foundation for further study of anti-inflammation functions and mechanisms of IL-37b.
ABSTRACT
@#<p style="text-align: justify;"><strong>BACKGROUND AND OBJECTIVES: </strong>IL-37b is a cytokine that may exist in several forms, including a full-length precursor protein and its putative mature forms (IL-37b cleaved at E21, 146, and K53, respectively). In recent years, the role of IL-37b has been associated with the regulation of inflammation and inflammatory diseases. Previous studies focused on the intracellular activity of the cytokine, while the bioactivities of its variants when introduced in the extracellular environment has been limited and require further investigation. To enable this, the study produced precursor and truncated forms of IL-37b in an E.coli expression system.</p><p style="text-align: justify;"><strong>METHODOLOGY:</strong> Recombinant proteins of the full-length (FL) and shorter forms (E21, 146, and K53) of IL-37b were produced in IPTG-induced E. coli BL21-CodonPlus(DE3)-RIPL strain and subsequently purified using Ni2+ NTA affinity, ion exchange, and size exclusion chromatography. The identity of the proteins was confirmed through western blotting and LC-MS.</p><p style="text-align: justify;"><strong>RESULTS:</strong> Findings showed that the masses of the expressed proteins correspond to their respective theoretical masses with 24,134.75 +0.04 Da for FL, 21,919.63 +0.80 Da for E21, 19,298.57 +0.04 Da for 146, and 18,551.21 +0.04 Da for K53 at 90-95% purity. This confirms that the correct proteins have been produced and at high purity. Further, the tendency of FL to homodimerize was observed in this study, which may have implications in the extracellular processing and bioactivity of FL.</p><p style="text-align: justify;"><strong>CONCLUSION:</strong> This study describes the successful expression and purification of recombinant precursor and putative mature forms of IL-37b in E.coli, which can be utilized for downstream characterization. </p>
Subject(s)
HumansABSTRACT
Objective To express the recombinant IL-37b protein and to investigate its role in the regulation of immune responses. Methods The prokaryotic expression vector pET28/IL-37b was constructed. The recombinant IL-37b protein was induced to be expressed and was purified using Ni2+-NTA gel column. C57BL/6 J mice were treated with IL-37b and injected with lipopolysaccharide(LPS)to induce septic shock,and the expression levels of IL-1β,IL-6,IFN-γ in the serum of the mice were detected. Dendritic cells from bone marrow of the mice were isolated and cultured, and were treated with IL-37b. After LPS-induced activation, the expression levels of marker molecules such as CD40, CD80 and MHCII on the cell surface, and cytokines such as IL-1β, IL-10, IL-12 and TNF-α in the culture supernatants were detected by flow cytometry. The CD4 +T cells from mice were isolated and the inhibitory effects of IL-37b on the expression of IFN-γ,TNF-α and IL-10 in the culture supernatants of T cells after induction by LPS were detected. Results IL-37b reduced the expression levels of proinflammatory cytokines in the serum of septic shock mice. IL-37b also inhibited the expression of co-stimulatory molecules and proinflammatory cytokines of the mouse dendritic cells, and suppress the activation of CD4 +T cells in vitro. Conclusions Purified recombinant IL-37b protein has high bioactivity, and can alleviate septic shock in organisms through inhibiting the activation of dendritic cells and related T cell immune responses.
ABSTRACT
Objective To investigate the expression of recombinant IL-37b protein and removal of the endotoxin, and identify its biological activity.Methods The prokaryotic expression vector pET28/IL-37b was constructed and to transform Escherichia coli (E.coli) Rosetta.After induction with IPTG, the recombinant protein was purified through Ni2+-NTA gel column and identified by SDS-PAGE and Coomassie brilliant blue staining.Then, the endotoxin protein was removed and was treated with LPS-stimulated RAW 264.7 cells.The culture supernatant was collected.The expression of IL-6 was detected by ELISA and the biological activity of the protein was identified.Results The recombinant IL-37b with high purity was expressed and the endotoxin produced by prokaryotic expression was reduced, and it was identified to have good biological activity.Conclusions In this study a recombinant IL-37b protein with high biological activity is successfully obtained.