塞内卡病毒A结构蛋白VP1诱导PK-15细胞凋亡  

The Structural Protein of Senecavirus A VP1 Induces Apoptosis in PK-15

作　　者：王咏 毛箬青[1] 张克山[1] 郑海学[1] 刘湘涛[1] WANG Yong;MAO Ruoqing;ZHANG Keshan;ZHENG Haixue;LIU Xiangtao(State Key Laboratory of Veterinary Etiological Biology,National Foot-and-Mouth Disease Reference Laboratory,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室国家口蹄疫参考实验室,兰州730046

出　　处：《畜牧兽医学报》2019年第4期811-820,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金重点项目(U1501213);云南省重点研发计划项目(2018BB004)

摘　　要：塞内卡病毒A(SVA)作为一种新发病病原,致病机制仍不清楚。通过显微镜观察发现,SVA感染PK-15细胞后能使细胞产生明显的细胞病变(CPE),同时伴随着严重的细胞凋亡。为了深入研究SVA诱导凋亡的情况,在验证SVA各蛋白真核质粒正常表达后,通过Annexin V-FITC/PI双染流式方法检测SVA各蛋白诱导凋亡的情况,Annexin V-FITC/PI和Hoechst染色后显微镜观察磷脂酰丝氨酸和核凝聚程度,通过Western blotting和RT-PCR技术分析SVA-VP1对凋亡通路中主要调控分子Caspase3、Caspase8和Bax蛋白质和mRNA水平的影响,发现SVA-VP1能够显著诱导细胞发生早期和晚期凋亡,并且能够促进凋亡蛋白Caspase3、Caspase8及Bax蛋白和mRNA水平的上调。本研究结果为深入研究SVA调控宿主细胞凋亡的分子机制和致病机制奠定了理论基础。As a new pathogen,the pathogenesis of Senecavirus A(SVA)remains unclear.Through microscopic observation,it was found that SVA can infect PK-15 cells and causes significant cytopathic(CPE).Concomitant with CPE,apoptosis is commonly observed in infected cells.In this study,the function of each individual SVA protein in the induction of apoptosis was studied.Firstly,using Western blotting confirmed the successful expression of SVA proteins.Subsequently,the SVA proteins were screened for their apoptotic function using flow cytometry with Annexin V-FITC/PI double staining.Then,the extent of externalized phospholipid phosphatidylserine and nuclear condensation induced by VP1 was analyzed by fluorescence microscope using Annexin V-FITC/PI and Hoechst staining.Finally,Western blotting and RT-PCR technique were used to analyze the effects of VP1 on the protein and mRNA levels of the major apoptosis regulatory molecules Caspase3,Caspase8 and Bax.SVA-VP1 was found to significantly induce early and late apoptosis in cells.And it also could promote the up-regulation of pro-apoptotic protein Caspase3,Caspase8 and Bax protein and mRNA levels.This study provides a theoretical basis for further research in molecular mechanism and the pathogenesis of SVA-regulated apoptosis.

关 键 词：塞内卡病毒A PK-15细胞 细胞凋亡 流式细胞术 

分 类 号：S852.659.6[农业科学—基础兽医学]