Construction of eukaryotic expression vector of SIGIRR-EGFP and its subcellular localization in H292 cells

Objective To analyze the expression and distribution of SIGIRR-EGFP in human air-way epithelial cells H292 using enhanced green fluorescent protein tracer technology and its impact on immune function of the cells.Methods A eukaryotic expression vector for single Ig IL-1R-related molecule(SIGIRR) fused with enhanced green fluorescent protein(EGFP) gene was constructed and transfected into H292.The subcellular localization of SIGIRR-EGFP was observed by laser confocal microscope.ELISA showed the change of IL-6 concentration in EGFP-transfected and SIGIRR-EGFP transfected H292 cells stimulated by LPS.Results The recombinant plasmid SIGIRR-EGFP was digested with EcoRI and BamHI,and the size of the bands were around 4.7 and 1.2 kb,respectively.The sequenced result of SIGIRR was the same as published sequence in GenBank.Expression of the fusion protein was mainly distributed in the intracellular compartments and had seldom distributed on the cell membrane.In contrast,only EGFP expression could be detected in H292 cells transfected with empty vector with diffuse distribution in the cells.The level of TNF-α was decreased significantly in SIGIRR-EGFP transfected H292 cells(P0.01) in comparison with untransfected and pEGFP-N1 transfected cells.Conclusion The expression vector for SIGIRR-EGFP fusion gene has been constructed and expressed in H292 cells successfully.The distribution characteristics of the expressed fusion protein is illustrated.Overexpression of SIGIRR can inhibit LPS-mediated native immune response.