摘要
采用PCR扩增长为1 041bp的山羊痘病毒(GTPV)serpin基因,构建真核表达质粒pcDNA-Ser-pin;将其转染HeLa细胞,经G418筛选,克隆纯化、鉴定,获得1株稳定表达GTPV SERPIN蛋白的HeLa细胞株;然后通过检测凋亡诱导剂TNF-α和放线菌酮作用后的细胞形态学变化、细胞活力和caspase活性,对GTPV SERPIN蛋白抑制宿主细胞凋亡的活性进行了鉴定。序列分析结果表明,GTPV SERPIN与正痘病毒SPI-1和SPI-2的氨基酸序列同源性为36%～38%;与痘苗病毒SPI-2空间构型相近,VADC基序和P1氨基酸(天门冬氨酸)相同。细胞试验结果显示,GTPV SERPIN蛋白能抑制由TNF-α和放线菌酮诱导的细胞凋亡,抑制率达70%左右,且这种抑制作用与其下调细胞caspase-8活性有关。证实GTPV SERPIN能抑制宿主细胞凋亡,可能是GTPV的致病因子之一。
Serpin gene（ORF149） of goatpox virus（GTPV） AV41 strain with length of 1 041 bp was amplified by PCR and subcloned into the expression plasmid pcDNA.A stable HeLa cell strain expressing GTPV SERPIN was established by transfecting pcDNA-Serpin into HeLa cell under selection of G418.After HeLa cell being treated with TNF-α and cycloheximide,cellular morphology observation,cell viability and caspase activity measure were undertaken to study the inhibition activity of GTPV SERPIN on host cell apoptosis.Results of sequence analysis showed that GTPV SERPIN shared 36% to 38% amino acid sequence homology with that of SPI-1 and SPI-2 of orthopoxviruses.Furthermore,GTPV SERPIN showed a similar molecular structure,conservative VADC motif and the same P1 amino acid-Asp（D） in reactive center loop with vaccinia virus SPI-2,implying they have similar bio-activity.Results of morphology observation,cell viability and caspase activity measure showed GTPV SERPIN could hamper the activation of caspase-8,and inhibit about 70% apoptosis of HeLa cell mediated by TNF-α and cycloheximide.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2013年第1期15-21,共7页
Chinese Veterinary Science
基金
广西青年科学基金项目(桂科青0991042)
