山羊痘病毒抗凋亡蛋白基因的表达与细胞凋亡的相关性

Expression of anti-apoptosis protein gene in goat pox virus and its relevance with the apoptosis of Vero cells

以贵州本地分离纯化的山羊痘病毒为试验材料,感染Vero细胞,通过台盼蓝、吖啶橙/溴乙锭荧光染色分析细胞的死亡及凋亡比例;经特异性PCR从山羊痘病毒基因组中分离出山羊痘病毒抗凋亡蛋白样基因,基因长531bp,含有完整的编码框,与绵羊痘病毒抗凋亡蛋白基因的相似性为97%,因此确定为山羊痘病毒抗凋亡蛋白基因。进一步研究山羊痘病毒抗凋亡蛋白基因在Vero细胞中的表达,结果显示,病毒感染Vero细胞24h时没有检测到凋亡现象,山羊痘病毒抗凋亡蛋白基因的mRNA水平最高;感染48h时,Vero细胞的凋亡率上升到13%,山羊痘病毒抗凋亡蛋白基因的表达量随之下降。结果表明,山羊痘病毒抗凋亡蛋白基因的表达量与Vero细胞的凋亡呈负相关,有助于病毒在细胞内的生存和侵染等过程。

To investigate the pathogenesis mechanism of goat pox virus（GPV） in goat pox,GPV strain LD isolated from the local goat in Guizhou province,China was chosen to infect sensitive Vero cells.The rates of death and apoptosis of Vero cells after infected by GPV were detected by trypan blue dye exclusion staining and acridine orange-ethidium bromide（AO/EB） fluorescent dye staining methods,respectively.The goatpox virus antiapoptosis protein-like gene was cloned from the genomic DNA of goatpox virus by special polymerase chain reaction（PCR）.The gene was 531 base pairs with similarity of 97% to the anti-apoptosis protein of sheep pox virus.Therefore the gene,named GGPVAP（goat pox virus anti-apoptosis protein）,was confirmed to be the anti-apoptosis protein gene of goat pox virus.The expression pattern of this gene was further analyzed.After infected with GPV for 24 hours,Vero cells didn′t show apoptosis but the expression level of GGPVAP gene was the highest.Forty-eight hours post infection,the apoptosis rate of Vero cells increased up to 13% while the expression of GGPVAP gene decreased rapidly.The expression of GGPVAP gene of goat pox virus was negatively correlated with the apoptosis rate of Vero cells,which suggested that the GGPVAP protein might inhibit the apoptosis of Vero cells to gain enough time for goat pox virus to finish its transcription and translation and invasion to other cells.