单纯疱疹病毒2型潜伏相关转录体ORF2的表达及其抗凋亡作用

The Expression and Anti-apoptotic Function of HSV-2 LAT ORF2

摘要:

本文探讨了单纯疱疹病毒2型 (HSV-2)潜伏相关转录体 (LAT)的开放读码框2 (ORF2)在细胞中的表达, 及其对5-氟尿嘧啶 (5-FU)诱导的非洲绿猴肾细胞 (Vero)凋亡的影响。通过将重组质粒pEGPF-ORF2转染Vero细胞, 绿色荧光蛋白检测转染效率, RT-PCR验证目的基因的表达, 5-FU 诱导细胞凋亡, 通过荧光显微镜观察凋亡小体, Gimesa染色检测细胞核形态, MTT法检测细胞的存活率, DNA ladder片段分析, 结果表明, 转染后绿色荧光蛋白表达效率很高, RT-PCR验证有目的基因的转录。凋亡诱导后的细胞形态正常, MTT法分析活性率与正常无差异, 而显著高于空质粒组, DNA ladder未见凋亡条带。由此我们认为HSV-2 LAT ORF2 基因在Vero细胞中得到了高效表达, 并且具有抗5-FU诱导的凋亡作用。

Abstract:

To explore the expression and effects of transfection of the HSV-2 LAT ORF2 gene on Vero cells against apoptosis induced by 5-FU. Recombinant plasmid pEGFP-ORF2 was transiently transfected into vero cells, expression was determined by green fluorescent protein and RT-PCR, changes of cell apoptosis were determined by Giemsa stain, MTT assay and DNA ladder. We found that the green fluorescent protein has a high expressed efficiency in Vero cells, target gene was detected by RT-PCR. Vero cells transfected with pEGFP-ORF2 induced by 5-FU have no changes in cytomorphology. MTT assay showed that the cells activ-ity transfected with HSV-2 LAT ORF2 remarkably higer than the negative control, While there were no such remarkable changes between them and normal cells. DNA ladder showed that there was no DNA fragment. Therefore we concluded that HSV-2 LAT ORF2 gene was expressed effectively in vero cells and can protect cells from apoptosis induced by 5-FU.