Construction and characterization of the recombinant expressive vector of human simplex virus 2-enhanced green fluorescent protein
- VernacularTitle:人单纯疱疹病毒加强型绿色荧光蛋白真核表达系统的构建及表达
- Author:
Leisheng LI
;
Hong YU
;
Wenqing ZHANG
- Publication Type:Journal Article
- Keywords:
Herpes simplex virus 2;
Green fluorescent protein;
ICP10 promoter
- From:
Journal of Medical Postgraduates
2003;0(07):-
- CountryChina
- Language:Chinese
-
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.
