携带高滴度猪瘟病毒C株猪睾丸细胞株的建立及蛋白质组学分析  

Establishment and Proteomics Analysis of Porcine Testis Cell Line Carrying Strain C of Swine Fever Virus with High-Titer

作　　者：张智慧 魏衍全 谷玲军 张韵[1] 孙世琪[1] 尹双辉[1] 郭慧琛[1,2] ZHANG Zhizhi;WEI Yanquan;GU Lingjun;ZHANG Yun;SUN Shiqi;YIN Shuanghui;GUO Huichen(StateKey Laboratory of Veterinary Etological Brology and Natonal Foot-and-Mouth Disease Reference Laboratory/Lanzhou VeternaryRsearch lnstiute,Chinese Academy of Agricultural Sciences,Lanzhou730046,China;College of animal Science,Yangze Unitversty,Jingzhou 434025,China;College of Veterinary Medicine,Gansu Agriculture Unversity,Lanzhou730070,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国家口蹄疫参考实验室,兰州730046 [2]长江大学动物科学学院,荆州434025 [3]甘肃农业大学动物医学院,兰州730070

出　　处：《病毒学报》2021年第3期648-657,共10页Chinese Journal of Virology

基　　金：国家重点研发计划项目(项目号:2017YFD0500906),题目:牛羊疫苗新型佐剂及工艺研究;国家自然科学基金项目(项目号:31672592),题目:仿生矿化优化口蹄疫病毒样颗粒耐热抗逆性研究。

摘　　要：猪睾丸细胞(Swine testicular,ST)广泛用于猪瘟病毒(Classical swine fever virus,CSFV)的传代,为了进一步提高ST细胞增殖的CSFV滴度,我们利用有限稀释法克隆出一种稳定携带高滴度CSFV C株的单克隆细胞株(STC)。间接免疫荧光实验结果显示,几乎100%STC细胞携带CSFV抗原;且STC细胞中CSFV滴度为106TCID_(50)/mL,与CSFV单次感染亲本ST细胞的病毒滴度10^(4)TCID_(50)/mL相比,具有更高的病毒产量。为了探究STC细胞持续带毒后细胞内蛋白表达水平的变化,我们通过非标记定量蛋白质组学分析发现541种蛋白表达水平发生明显变化,并选取其中差异显著且与病毒复制或细胞存活相关的两种上调蛋白(GRP94,GRP78)、两种下调蛋白(β-catenin,ISG15)进行免疫印迹验证。本试验成功获得了可以稳定携带高滴度CSFV C株的STC细胞株,并初步筛选了与细胞持续带毒有关的目标蛋白,不仅为简化猪瘟传代细胞苗生产工艺提供条件,也为研究STC细胞持续携带CSFV稳定传代的机制奠定基础。Swine testicular cells(ST)are widely used for the passage of classical swine fever virus(CSFV).To further increase the virus titer in ST cells,we applied the limited dilution method to clone a homogeneous monoclonal cell line stably carrying high titer of CSFV strain C,named STC.Indirect immunofluorescence experiments revealed that almost 100%of STC cells carried CSFV antigen.The CSFV titers in STC cells was up to 106 TCID50/mL,which has higher virus yield compared with the virus titer in CSFV infected parent ST cells(104 TCID50/mL).Then the label-free quantitative proteomic analysis was performed to explore the changes in protein expression of STC cells.Among 541 proteins with significantly altered expression,two proteins with up-regulated expression(GRP94,GRP78)and two proteins with down-regulated expression(β-catenin,ISG15)related to viral replication or cell survival were selected for immunoblotting verification.Our data suggest that we obtained STC monoclonal cell lines that could stably carry high titers of CSFV strain C,and initially screened the target proteins related to persistent infection.This approach will not only simplify subculture of CSFV seedlings,but also lay the foundation for studying the mechanisms of persistent carriage of CSFV in STC cells.

关 键 词：猪瘟病毒C株 稳定细胞株 高滴度带毒 蛋白表达差异 

分 类 号：S852.651[农业科学—基础兽医学]