ABSTRACT
Objective To synthesize herpes simplex virus (HSV) envelope glycoprotein gC using gene engineering techniques,and to verify its expression.Methods Two separate parts of the HSV envelope glycoprotein gC,i.e.,GC-F and GC-R,were respectively synthesized.The GC-F and GC-R genes were synthesized,subcloned into the expression vectors pSumo-Mut (containing recognition sequences for endonucleases Stu1 and XhoI) and pCzn1 (containing recognition sequences for endonucleases NdeI and XhoI) respectively to form the recombinant plasmids pSumo-Mut-GC-F and pCzn1-GC-R.E.coli BL21 Arctic Express (DE3) cells were transformed with the two recombinant plasmids separately.Isopropyl thiogalactoside (IPTG) was used to induce the expression of target protein which was subsequently purified by nickel affinity chromatography.Finally,Western blot was performed to verify the reactivity of the synthesized protein with the sera of HSV-1-positive patients.Results Both GC-F and GC-R genes were synthesized by a total gene synthesis method.As sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) showed,the fusion proteins were mainly distributed in the sediment layer.The purity of GC-F and GC-R proteins was over 80％ after purification by affinity chromatography.Western blot showed that both of the proteins were reactive with anti-HSV-1 antibody-positive sera.Conclusions Fusion expression vectors have been constructed for the gC protein,and IPTG successfully induces its expression.Moreover,the resulting proteins could react with anti-HSV-1 antibody-positive sera,and may serve as an ideal experimental material for next functional study.
ABSTRACT
Objective To establish a cellular model for the expression of the C-type lectin dendritic cellspecific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN),and to provide a basis for the functional analysis of DC-SIGN.Methods The cDNA of DC-SIGN was obtained via PCR,and cloned into the eukaryotic expression vector porcine cytomegalovirus-enhanced green fluorescent protein (PCMV-EGFP) with EGFP at the N terminal of DC-SIGN.Then,the recombinant PCMV-EGFP-DC-SIGN plasmid was transfected into HEK293T cells followed by the detection of DC-SIGN expression using PCR,Western blot and flow cytometry.Confocal microscopy was performed to localize the expression of DC-SIGN-EGFP and visualize the recognization and internalization of the Derp2 allergen by DC-SIGN.Results The recombinant fluorescent fusion protein-expressing plasmid was successfully constructed.Both PCR and Western blot confirmed the expression of DC-SIGN.Flow cytometry showed that the expression of DC-SIGN was increased by approximately 50％ in HEK293T cells transfected with the recombinant expression plasmid compared with those untransfected.As confocal microscopy showed,the green fluorescence-labelled DC-SIGN was located on the cell membrane,which could bind to the red fluorescence-labelled antigen Derp2 and internalize it into the cells.Conclusions The recombinant DC-SIGN-EGFP fusion protein is characteristically located on the surface of 293T cells,which can be recognized by the DC-SIGN-specific antibody and is capable of internalizing the allergen Derp2,and may serve as an ideal cell model for further studies on molecular function of DC-SIGN.