Establishment Of The Cell Line For Detection Of Herpes Simplex Virus Based On Enhanced Green Fluorescent Protein

Objective To establish the cell line for detection of herpes simplex virus based on enhanced green fluorescent protein.Methods A pair of PCR primers were created from the ICP10 promoter gene of HSV-2 (Sal 5 strain) by DNA Star software and two restriction sites (Bgl II and Hind HI) were included as part of the primers. The gene fragment coding for ICP10 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 pICP10-EGFP, which would be identified by enzyme digestion and DNA sequencing. The recombinant plasmid pICP10-EGFP were transfected into Vero cells by cation lipoid Lipofectamine2000. 48h after the transfection, DMEM culture medium containing G418(400 μg/ml) was added to screen the positive cells to obtain stable cell line Vero-ICPlO-EGFP. Vero-ICPlO-EGFP was infected by various titers of HSV-2, and the GFP fluorescence was detected under inverted microscope at 6, 8 and 10h post-infection.Results A 641bp fragment was amplified by PCR. The recombinant plasmid pICPlO-EGFP was proved to correct by DNA sequencing. The positive cells were isolated by G418 lldays post-transfection. The Vero-ICP10-EGFP fluorescent emitting cells can be observed as early as 6h after infection with HSV, with pronounced increase in the intensities at later hours.Conclusion The establishment of Vero-ICP10-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.