稳定表达绿色荧光蛋白标记的人单纯疱疹病毒2型潜伏相关转录体ORF2融合蛋白的Vero细胞株的建立

Establishment of Vero Cell Strain for Stable Expression of Fusion Protein of Open Reading Frame 2 of Human Herpes Simplex Virus Type 2 Latency Associated Transcript and Enhanced Green Fluorescent Protein

目的建立稳定表达绿色荧光蛋白(EGFP)标记的人单纯疱疹病毒2型(HSV-2)潜伏相关转录体(LAT)开放读码框2(ORF2)融合蛋白的Vero细胞株。方法构建重组真核表达质粒pEGFP-ORF2,经酶切及测序鉴定正确后,体外转染Vero细胞,经G418筛选稳定表达融合蛋白的克隆,扩大培养后,荧光显微镜观察EGFP的表达,RT-PCR检测目的基因的转录。结果重组真核表达质粒pEGFP-ORF2经酶切及测序鉴定构建正确,经G418培养20d筛选出的Vero细胞株荧光显微镜下可见融合蛋白表达,RT-PCR检测显示,转染了重组表达质粒的Vero细胞内有目的基因的表达。结论已成功建立了稳定表达EGFP-ORF2的Vero细胞株,为进一步研究HSV-2LATORF2的功能奠定了基础。

Objective To establish a Vero cell strain for stable expression of fusion protein of open reading frame 2（ORF2） of human herpes simplex virus type 2（HSV-2）latency associated transcript（LAT）and enhanced green fluorescent protein（EGFP）.Methods Recombinant plasmid pEGFP-ORF2 was constructed and identified by restriction analysis and sequencing,then transfected to Vero cells in vitro.The recombinants for stable expression of fusion protein were screened with G418 for scale-up culture,in which the expression of EGFP was observed by fluorescent microscopy,and the transcription of target gene by RT-PCR.Results Both restriction analysis and sequencing proved that recombinant plasmid pEGFP-ORF2 was constructed correctly.The expression of fusion protein was proved by fluorescent microscopy in the screened Vero cell strain.RT-PCR showed transcription of target gene in Vero cells transfected with recombinant plasmid pEGFP-ORF2.Conclusion A Vero cells strain for stable expression of fusion protein of HSV-2 LAT ORF2 and EGFP was successfully established,which laid a foundation of further study on function of HSV-2 LAT ORF2.