论文部分内容阅读
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 line stably expressing the green fluorescent protein (EGFP) tagged human herpes simplex virus type 2 (HSV-2) LAT open reading frame 2 (ORF2) fusion protein. Methods The recombinant eukaryotic expression plasmid pEGFP-ORF2 was constructed and confirmed by restriction enzyme digestion and sequencing. The recombinant plasmid was transfected into Vero cells in vitro and cloned by G418. The expression of EGFP was observed by fluorescence microscopy. RT-PCR Detection of target gene transcription. Results The recombinant plasmid pEGFP-ORF2 was identified by restriction enzyme digestion and sequencing. The fusion protein was expressed under fluorescence microscope in Vero cell line selected by G418 for 20 days. RT-PCR showed that Vero cells transfected with recombinant plasmid The purpose of gene expression in the cell. Conclusion The Vero cell line stably expressing EGFP-ORF2 has been successfully established, which lays the foundation for further study on the function of HSV-2LATORF2.