Construction and characterization of the recombinant expressive vector of human simplex virus 2-enhanced green fluorescent protein / 医学研究生学报

ABSTRACT

Objective: To construct the recombinant expressive vector of HSV-2-EGFP for the primary application in rapid,direct and sensitive diagnosis of herpes simplex virus infection. Methods: The gene fragment coding for HSV-2 ICP10 promoter was amplified by PCR,and the PCR product was cloned into pEGFP-1 to construct the recombinant plasmid pICP10-EGFP,which would be identified by DNA sequencing.The recombinant plasmid pICP10-EGFP were transfected into Vero cells by cation lipoid Lipofectamine 2000,then G418 was added to screen the positive cells to obtain stable cell line Vero-ICP10-EGFP.Vero-ICP10-EGFP was infected by various titers of HSV-2,and the EGFP fluorescence was detected under inverted microscope at 6,8 and 10 h post-infection.The specificity of the cell line was detected by infection with human cytomegalovirus(HCMV) and coxsackievirus. Results: The recombinant plasmid pICP10-EGFP was proved to be correct by the double restriction enzyme digestion and DNA sequencing.The Vero-ICP10-EGFP fluorescent emitting cells can be observed as early as 6 h after infection with HSV,with pronounced increase in the intensities at later hours.No induction of detectable fluorescence was detected by infection with HCMV and coxsackievirus 6,10 and 24 h post-infection. Conclusion: This novel GFP reporter system expressing the HSV-inducible EGFP reporter gene might provide a fast,easy and sensitive model for monitoring HSV in clinical specimens.