猪繁殖与呼吸综合征病毒N蛋白基因的克隆及表达  

Cloning and expression of nucleocapsid protein gene of porcine reproductive and respiratory syndrome virus(PRRSV)

作　　者：刘斌[1,2] 才学鹏[2] 郭爱疆[2] 贾万忠[2] 骆学农[2] 陈晓宇[1,2] 刘振勇[2] 丁军涛[2] 刘英[1] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]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,甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《甘肃农业大学学报》2009年第1期27-31,共5页Journal of Gansu Agricultural University

基　　金：国家高科技研究发展计划“863”项目(2006AA10A207);甘肃省重大科技专项(2GS063-A43-013)

摘　　要：根据猪繁殖与呼吸综合征病毒(PRRSV)核酸序列设计引物,用RT-PCR扩增出PRRSV甘肃株的核衣壳蛋白(N)基因,将N基因克隆至pGEM-T easy载体上,获得含N基因的阳性重组质粒pGEM-T easy-N.对质粒中的插入片段进行测序,应用DNAStar分析所测序列与GenBank中已登录的PRRSV毒株的同源性,再将其亚克隆至原核表达载体pGEX-4T-1中,得到重组表达载体pGEX-4T-1-N,转化E.coliBL21(DE3)细胞,用SDS-PAGE检测表达产物,并对其进行纯化.结果表明:PRRSV甘肃株N基因核甘酸序列与PRRSV-VR2332株的同源性为93.3%,与欧洲LV株的同源性为66.1%;构建的重组表达载体在大肠杆菌中获得了成功表达,表达产物为40ku的可溶性融合蛋白.Porcine reproductive and respiratory syndrome virus （PRRSV） nucleoprotein gene,amplified by reverse transcription-PCR was cloned into pGEM-T easy vector, After sequencing, the N gene was blasted with PRRSV genome sequence downloaded from GenBank with DNAstar software. The results indicated that the homologus nucleotide sequence of N of PRRSV-GS was 93.3 % and 66. 1 % compared with PRRSV-VR2332 and LV. The N gens was subcloned into prokaryotic expressing vector pGEX-4T-1, the recombinant plasmid named pGEX-4T-1-N was constructed. The pGEX-4T-1-N was used to transform into E. coli BL21 （DE3）. The results of SDS-PAGE showed that the N gene was expressed in a high level as a 40 ku .

关 键 词：猪繁殖与呼吸综合征病毒(PRRSV) N基因 克隆与表达 

分 类 号：S852.65[农业科学—基础兽医学]