Expression of pcDNA3-TLR9/GFP in HEK293T cells

Objective To construct a plasmid encoding the human Toll-like receptor 9(TLR9) full length and green fluorescent protein(GFP),and investigate the expression of TLR9/GFP fusion protein in HEK293T cells in order to detect its response to CpG DNA stimulation.Methods According to the sequence of human TLR9 gene recorded in GenBank,primers of TLR9 full length were designed.The objective gene fragment was amplified by PCR,digested by BamHⅠ and XhoⅠ,and cloned into pcDNA3/GFP plasmid.After being identified by enzyme digestion and sequencing,the recombinant vector was transfected into HEK293T cells.The green fluorescence and cellular localization of TLR9/GFP fusion protein were observed by fluorescent microscopy.Meanwhile,the luciferase activity of HEK293T co-transfected with pcDNA3-TLR9/GFP and pGL2-luc(NF-κB-luciferase reporter) following CpG DNA stimulation were measured by using luciferase reporter assay.Results The recombinant pcDNA3-TLR9/GFP had been confirmed to be correct constructed after sequencing identification.Following successful transfection to HEK293T with the plasmid,the green fluorescent TLR9/GFP fusion protein was expressed in cells.The luciferase activity of HEK293T co-transfected with pcDNA3-TLR9/GFP and pGL2-luc was increased significantly after stimulated with CpG DNA(10 mg/L).Conclusion The recombinant pcDNA3-TLR9/GFP is constructed successfully and the TLR9/GFP fusion protein which can respond to CpG DNA stimulation were expressed in HEK293T.