猪繁殖与呼吸综合征病毒NSP12蛋白的表达与鉴定  

PROKARYOTIC EXPRESSION AND IDENTIFICATION OF NON-STRUCTURAL PROTEIN 12 OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS

作　　者：张文超[1,2] 虞凌雪[2] 姜一峰[2] 高飞[2] 黄勤峰[2] 李丽薇 李慧春[2] 陈鹏飞[2] 杨德强[2] 刘欢[2] 童光志[2] 杜雅楠[1] 周艳君[2] 

机构地区：[1]内蒙古农业大学兽医学院,呼和浩特010018 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2018年第1期32-36,共5页Chinese Journal of Animal Infectious Diseases

基　　金：973计划项目(2014CB542702);国家科技支撑计划课题(2015BAD12B01-1);国家生猪现代产业技术体系项目(CARS-36)

摘　　要：本研究扩增了高致病性猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)Hu N4株的NSP12基因,将其克隆于pCold-Ⅰ载体中,获得重组质粒pCold-Ⅰ-NSP12,并转化于E.coli/BL21感受态细胞,在16℃条件下用1 mmol/L的IPTG诱导20 h后,用SDS-PAGE电泳检测表达产物,结果显示在大约18 kDa处出现目标蛋白。用PRRSV特异性阳性猪血清对表达蛋白进行Western blot鉴定,结果显示该蛋白可以被PRRSV阳性猪血清识别。将重组NSP12蛋白纯化后,免疫6周龄BALB/c小鼠制备多克隆抗体。在MARC-145细胞中,对获得的NSP12多抗血清分别进行Western blot和间接免疫荧光检测(indirect immunofluorescence assay,IFA)鉴定,结果显示本研究制备的NSP12多抗血清均可与感染的MARC-145细胞的PRRSV Hu N4株以及在MARC-145细胞中特异表达的NSP12蛋白发生免疫反应。PRRSV NSP12蛋白的表达及其多克隆抗体的制备为进一步研究NSP12在病毒感染过程中的作用奠定了基础。The non-structural protein12 （NSP12） of Porcine reproductive and respiratory syndrome virus （PRRSV） was amplifled and cloned into the pCold-I vector. The recombinant plasmid pCold-Nsp12 was then transformed into E.coli /BL21 cells and induced with 1 mmol/L isopropyl β -D-thiogalactoside （IPTG） at 16℃ for 20 h. The recombinant protein was purified and identified with SDS-PAGE as about 18kDa band and also recognized in Western blotting using PRRSV antisera. Subsequently, the purified recombinant Nsp12 protein was administered into 6-week-old BALB/c mice for production of polyclonal antibodies. The antisera collected from these immunized mice speciflcally reacted with PRRSV HuN4 strain and NSP12 protein expressed in MARC-145 in IFA and Western blotting. The present study laid a basis for further study on the role of NSP12 in PRRSV infection.

关 键 词：猪繁殖与呼吸综合征病毒 NSP12 原核表达 多抗 

分 类 号：S852.659.6[农业科学—基础兽医学]