稳定表达山羊SLAM受体的BHK-21细胞系的建立及应用  

The establishment and application of BHK-21 expressing goat SLAM

作　　者：张家林[1] 李翠翠[1] 谢梅梅 王子龙[1] 陈伟业[1] 步志高[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2015年第7期506-509,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家科技重大专项(2012ZX1000214);国家高科技研究发展计划(863计划)(2011AA10A208);国家科技支撑计划课题:严重威胁的外来动物疫病免疫防控储备技术(2013BAD12B05)

摘　　要：信号淋巴激活分子(SLAM)又称为CD150,是小反刍兽疫病毒(PPRV)和犬瘟热病毒(CDV)等麻疹病毒属病毒感染淋巴细胞的主要受体,在病毒侵入细胞中发挥重要作用。为建立稳定表达山羊SLAM(g SLAM)的真核细胞系,本研究将人工合成的g SLAM基因克隆至真核表达质粒p IRESpuro3中,并在该基因的3'端引入Flag标签序列作为分子标记,构建了重组质粒p IRES3-g SLAM。将该重组质粒转染BHK-21细胞,经嘌呤霉素加压筛选及采用表达绿色荧光蛋白(GFP)的重组PPRV病毒(r PPRV/GFP)感染鉴定后,筛选到稳定表达g SLAM基因的细胞系。r PPRV/GFP感染和western blot鉴定表明,无论是否有嘌呤霉素压力的存在,该细胞系在传代至第20代,仍能稳定表达g SLAM蛋白。由于g SLAM氨基酸序列与犬的同源性较高,以表达GFP重组CDV强毒株(r CDV/GFP)感染该细胞系,病毒可以感染且能形成明显的细胞病变,表明该细胞系可用于CDV强毒分离和致弱机制等相关研究。Signaling lymphocyte activation molecule（SLAM or CD150） is the major receptor for Morbillivirus such as peste des petits ruminants virus（PPRV） and canine distemper virus（CDV） to infect the lymph cells. In order to establish the cell line stably expressing goat SLAM（g SLAM）, the g SLAM gene was synthetized artificially and cloned into the p IRESpuro3 to generate the recombinant plasmid of p IRES3-g SLAM. Then, the cell line stably expressing g SLAM（BHK-g SLAM） was established by transfection of BHK-21 cell with p IRES3-g SLAM screening under puromycin pressure and identified by infection with recombinant PPRV expressing GFP（r PPRV/GFP）. In addition, the cell line stably expressing g SLAM at 20 passages either with or without the puromycin selection pressure verified by r PPRV/GFP infection test and western blot. Furthermore, the recombinant virulent CDV expressing GFP was also able to infect the BHK-g SLAM and replicated in the cell line detected by GFP expression.These results suggested that the BHK-g SLAM was a permissive cell line for both r PPRV and CDV, which would facilitate future study of the viral infection mechanism.

关 键 词：信号淋巴细胞激活分子 BHK-21细胞 小反刍兽疫 犬瘟热 病毒分离 

分 类 号：S852.65[农业科学—基础兽医学]