Eukaryotic Expression Of Human Simplex Virus 1 Green Fluorescent Protein And Its Primary Application

Objective To construct the recombinant expressive vector of HSV-1-GFP for the primary application in rapid, direct and sensitive diagnosis of herpes simplex virus infection.Methods A pair of PCR primers were created from the ICP6 promoter gene of HSV-1 SM44 strain by DNA Star software and two restriction sites (BamH I and Hind III) were included as part of the primers. The gene fragment coding for ICP6 promoter was amplified by PCR. The PCR product was subcloned into plasmid pMD18-T and then cloned into pEGFP-1 to construct the recombinant plasmid pICP6-EGFP, which would be identified by enzyme digestion and DNA sequencing. The recombinant plasmid pICP6-EGFP were transfected into Vero cells by cation lipoid Lipofectamine2000. 48h after the transfection, DMEM culture medium containing G418(400 g/ml) was added to screen the positive cells to obtain stable cells line Vero-ICP6-EGFP. Vero-ICP6-EGFP was infected by various titers of HSV-1, and the GFP fluorescence was detected under inverted microscope at 6, 8 and 10h post-infection. The intensity of fluorescence was measured by flow cytometry 15h post-infection. The specificity of the cell line was detected by infection with human cytomegalovirus (HCMV) and coxsackievirus. Results A 441bp fragment was amplified by PCR. The recombinant plasmid pICP6-EGFP were proved to correct by the double restriction enzyme digestion and DNA sequencing. The positive cells were isolated by G418 11 days post transfection. The Vero-ICP6-EGFP fluorescent emitting cells can be observed as early as 6h after infection with HSV, with pronounced increase in the intensities at later hours. Analysis by flow cytometry also demonstrated that intensity of the triggered fluorescence is proportional to the titer of HSV inoculated. No induction of detectable fluorescence was detected by infection with HCMV and coxsackievirus 6h, 10h and 24h post-infection. Conclusion The establishment of Vero-ICP6-EGFP cell line expressing the HSV-inducible EGFP reporter gene might provide a fast, easy and sensitive model for monitoring HSV in clinical specimens. This novel GFP reporter system could become a useful means for screening antiviral drug.