单纯疱疹病毒2型潜伏相关转录体ORF1的表达及其抗凋亡作用

Expression of herpes simplex virus type 2 latency associated transcript ORF1 and its anti-apoptotic function

摘要:

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-2 333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。

关键词:单纯疱疹病毒，LAT，ORF1，放线菌素D，凋亡，Veo细胞

Abstract:

To study the expression of herpes simplex virus type 2 latency-associated transcript (LAT) open reading frame 1 (ORF1) and its anti-apoptosis function induced by actinomycin D in Vero cells. The recombinant plasmid pEGFP-ORF1 was constructed and transfected into Vero cells, and the expression of ORF1 was identified by RT-PCR. The changes of Vero cells morphology induced by actinomycin D were observed by fluorescence microscopy, Hochest33258 fluorescence staining. Cells viability was evaluated by MTT assay and cells apoptosis rate was detected by flow cytometry. Double digestion and sequencing confirmed the pEGFP-ORF1 was constructed successfully, RT-PCR showed that the target gene was highly expressed in Vero cells. Hochest33258 staining reaveals that Vero cells transfected with pEGFP-ORF1 and induced apoptosis by actinomycin D had no changes in morphology. MTT assay showed that the viabilities of Vero cells transfected with recombinant plasmid pEGFP-ORF1 and induced apoptosis by actinomycin D has no statistically significant difference compared with the untreated normal control group (P>0.05), but remarkable higher than Vero cells transfected with empty plasmid pEGFP-C2 and induced apoptosis by actinomycin D, the difference was statistically significant (P<0.05). Flow cytometry assay shows that the cells apoptosis rate had no significant difference between pEGFP-ORF1 group and the normal group, but the cells apoptosis rate of pEGFP-ORF1 was lower than the pEGFP-C2 group. HSV-2 LAT ORF1 gene can be expressed in Vero cells and can protect Vero cells from apoptosis induced by actinomycin D.

Key words:herpes simplex virus, LAT, ORF1, actinomycin D, apoptosis, Vero cells