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旨在研究单纯疱疹病毒2型潜伏相关转录体(LAT)开放读码框1(ORF1)对放线菌素D诱导的凋亡作用的影响。以HSV-2333基因组为模板PCR扩增ORF1片段,构建重组质粒pEGFP-ORF1,转染Vero细胞,RT-PCR鉴定ORF1的表达。放线菌素D诱导Vero细胞凋亡,通过荧光显微镜观察凋亡小体,Hochest33258荧光染色观察细胞形态变化,MTT检测细胞活性,流式细胞术检测细胞凋亡率。双酶切和测序确认pEGFP-ORF1构建成功,RT-PCR表明该真核表达载体能在Vero细胞中高效表达。转染了pEGFP-ORF1的Vero细胞经放线菌素D凋亡诱导后,Hochest33258染色显示细胞形态正常。MTT结果表明转染了重组质粒pEGFP-ORF1的Vero细胞经放线菌素D凋亡诱导后Vero细胞活性与未经任何处理的正常对照组相比,无显著差异(P>0.05),但高于放线菌素D诱导凋亡的Vero细胞组及与转染空质粒pEGFP-C2且放线菌素D诱导凋亡的Vero细胞组,差异具有统计学意义(P<0.05)。流式结果表明,转染重组质粒pEGFP-ORF1且经放线菌素D诱导凋亡组与正常对照组凋亡率差异不显著(P>0.05),而显著低于放线菌素D诱导凋亡组和转染空质粒pEGFP-C2且经放线菌素D诱导凋亡组(P<0.05)。HSV-2 LAT ORF1具有抗放线菌素D诱导的Vero细胞的凋亡作用。
The aim of this study was to investigate the effect of open reading frame 1 (ORF1) of herpes simplex virus type 2 (LAT) on actinomycin D-induced apoptosis. The ORF1 fragment was amplified by PCR using the HSV-2333 genome as a template. The recombinant plasmid pEGFP-ORF1 was constructed and transfected into Vero cells. The expression of ORF1 was identified by RT-PCR. Actinomycin D induced the apoptosis of Vero cells. The apoptotic bodies were observed by fluorescence microscopy. Hochest33258 fluorescence staining was used to observe the morphological changes of the cells. The cell viability was detected by MTT assay and the apoptosis rate by flow cytometry. Double-digestion and sequencing confirmed the successful construction of pEGFP-ORF1, and RT-PCR indicated that the eukaryotic expression vector could be efficiently expressed in Vero cells. Vero cells transfected with pEGFP-ORF1 were induced by actinomycin D, and Hochest33258 staining showed normal cell morphology. MTT assay showed that Vero cells transfected with recombinant plasmid pEGFP-ORF1 had no significant difference (P> 0.05) in the activity of Vero after induced by actinomycin D (P> 0.05) The Vero cells induced by actinomycin D and Vero cells transfected with empty plasmid pEGFP-C2 and actinomycin D had statistical significance (P <0.05). The results of flow cytometry showed that there was no significant difference in apoptosis rate between the group of pEGFP-ORF1 transfected with actinomycin D and the control group (P> 0.05), but significantly lower than that of the actinomycin D The apoptotic cells were transfected with empty plasmid pEGFP-C2 and induced by actinomycin D (P <0.05). HSV-2 LAT ORF1 has anti-actinomycin D-induced apoptosis in Vero cells.