建立以EGFP为报告基因检测HSV感染的细胞株

Establishment of the Cell Line for Detection of Herpes Simplex Virus Based on Enhanced Green Fluorescent Protein

通过PCR扩增HSV-1ICP6启动子目的基因片段,克隆至真核表达载体pEGFP-1构建重组质粒pICP6-EGFP,转染Vero细胞,经G418筛选获得稳定转染细胞株Vero-ICP6-EGFP细胞。以不同量的HSV-1感染Vero-ICP6-EGFP细胞,分别以倒置荧光显微镜和流式细胞术检测EGFP荧光的量和强度。结果表明,转染细胞株Vero-ICP6-EGFP感染HSV-16h后,倒置荧光显微镜下即可检测绿色荧光;流式细胞术检测结果表明,EGFP荧光的量及强度随HSV-1病毒的滴度增加而增加。已建立的以EGFP为报告基因的Vero-ICP6-EGFP细胞株具有早期、快速、灵敏等优点,特异性较高,可望用于HSV感染的快速诊断及抗病毒药物的检测。

The gene fragment coding for ICP6 promoter was amplified by PCH and the PCR product was 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, then G418 （400μg/mL） was added to screen the positive cells to obtain stable cell line Vero- ICP6-EGFP. Vero-ICP6-EGFP was infected by various titers of HSV-1, and the EGFP fluorescence was detected under inverted microscope at 6, 8 and 10h post-intection. The intensity of fluorescence was measured by flow cytometry 15h post-infection. The results showed that the recombinant plasmid pICP6-EGFP was proved to correct by the double restriction enzyme digestion and DNA sequencing. 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. It can be concluded that 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.