口蹄疫病毒VP1蛋白诱导犊牛甲状腺初代细胞凋亡的鉴定

Apoptosis induced by VP1 protein of foot-and-mouth disease virus in primary bovine thyroid cells

为了制备和培养犊牛甲状腺初代细胞(CTY),并检测口蹄疫病毒VP1蛋白对其诱导的凋亡情况,采用原代细胞培养方法,取初生犊牛甲状腺制备初代细胞并培养。采用RT-PCR方法扩增口蹄疫病毒VP1基因,再用限制性内切酶EcoRⅠ+XhoⅠ酶切后定向克隆到表达载体pCAGGS中,构建重组质粒pCAGGS-VP1,转染CTY,用Western-blot检测口蹄疫病毒VP1蛋白在犊牛甲状腺初代细胞中的表达情况;通过显微镜观察转染的CTY细胞状态、经AV-PI双染色、检测线粒体膜电位变化和Hoechst-33258荧光染色来检测细胞凋亡。结果显示,成功制备了犊牛甲状腺初代细胞,构建的重组质粒可以在犊牛甲状腺初代细胞中表达,通过几种方法均证明口蹄疫病毒VP1蛋白能够诱导犊牛甲状腺初代细胞的凋亡,细胞凋亡率比空载体对照组高约2倍。结果表明,口蹄疫病毒VP1蛋白能够诱导CTY的凋亡,这一结果为深入研究口蹄疫病毒VP1蛋白凋亡功能域和VP1诱导的凋亡途径奠定了基础。

The aim of this study was to prepare and culture the primary calf thyroid cells(primary calf thyroid,CTY),and to detect apoptosis induced by foot-and-mouth disease virus(FMDV)VP1 protein in primary calf thyroid cell.According to the protocol,the primary bovine thyroid cells were prepared and cultured.The VP1 gene of FMDV was amplified by RT-PCR and subcloned into expression vector pCAGGS by restriction endonuclease EcoRⅠand XhoⅠ,and the recombinant plasmid was confirmed by sequencing and restriction endonuclease digestion.The VP1 protein expression of pCAGGS-VP1 was analyzed by Western-blotting.The recombinant plasmid was transfected into CTY and then observed by fluorescence microscopy.Apoptosis was detected by AV-PI staining,mitochondrial membrane potential change and Hoechst-33258 fluorescent staining.The primary calf thyroid cells were successfully cultured,and the VP1 protein of FMDV was expressed in CTY and could induce cell apoptosis.The apoptosis induced by FMDV VP1protein in primary calf thyroid cells was confirmed.The results and data laid the foundation for studying apoptosis-associated functional domains in VP1protein and apoptotic pathways induced by FMDV VP1 protein.