小反刍兽疫病毒受体细胞系的构建及应用  

Construction and application of receptor cell line of peste des petits ruminants virus

作　　者：金红岩[1,2] 封家旺[2] 梁琳[1] 赵玲娜[1] 隋修锟[1] 史利军[1] 侯绍华[1] 李刚[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所农业部兽用药物与兽医生物技术北京科学观测实验站,北京100193 [2]西藏职业技术学院,西藏拉萨850030

出　　处：《中国兽医科学》2017年第8期1022-1026,共5页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(31172342);国家科技支撑计划项目(2013BAD12B05);国际合作项目(D32025/17453)

摘　　要：为建立一株稳定表达山羊淋巴细胞活化分子(signaling lym phocyte activation molecule,SLA M)的细胞系,给研究小反刍兽疫病毒(peste des petits ruminants virus,PPRV)感染机制、病毒的分离和疫苗生产等奠定基础,本研究采用PCR扩增了SLAM基因,并将其胞外区连接至pD isplay载体,构建了重组质粒pDisplayg SLAM;利用p Display载体的G 418抗性及抗SLAM单克隆抗体,筛选表达gSLAM的Vero-E6细胞系,对该细胞系进行PCR鉴定、间接免疫荧光试验和Western-blot鉴定。采用该细胞系分离PPRV,并与Vero-E6细胞同时吸附PPRV分离株和疫苗株病毒后进行比较。结果,成功筛选出表达g SLAM的Vero-E6细胞系gSLAM/Vero-E6,并通过PCR方法成功扩增出gSLAM基因。通过间接免疫荧光试验和Western-blot鉴定,gSLAM基因成功表达在细胞膜上,并分离出一株PPRV。gSLAM/Vero-E6细胞系和Vero-E6细胞同时吸附PPRV分离株和疫苗株后,gSLAM/Vero-E6细胞系较Vero-E6细胞产生更明显的细胞病变。上述研究结果表明,gSLAM/Vero-E6细胞系对PPRV是高度敏感,为PPRV的后续研究提供了试验材料。To establish cell lines with a stable expression of signaling lymphocyte activation molecule of goat,and to research the PPRV infection mechanisms,separation of virus and vaccine production,the extracellular domain of SLAM gene was amplified by PCR,then inserted into the p Display vector and named p Display-g SLAM.G418 and anti-SLAM monoclonal antibody were used to screen the Vero-E6 cell lines that could stably express g SLAM.The cell lines were identified by PCR,indirect immunofluorescence test and Western-blot.At the same time,PPRV virus was isolated by this cell line,and this cell line was absorbed by the PPRV isolate and vaccine strains together with Vero-E6 cells.In result,g SLAM/Vero-E6 was successfully screened,and the g SLAM gene was successfully amplified by PCR method.The g SLAM gene was successfully expressed on the cell membrane by indirect immunofluorescence and Western-blot,and the PPRV was isolated. At the same time,the adsorption of PPRV isolates and vaccine strains,compared with Vero-E6 cells,g SLAM/Vero-E6 cell lines had more obvious pathological changes.In conclusion,g SLAM/Vero-E6 cells are highly sensitive to PPRV in clinical specimens and provides experimental materials for follow-up studies of PPRV.

关 键 词：小反刍兽疫病毒 pDisplay载体 细胞系 

分 类 号：S858.2[农业科学—临床兽医学]