猪生殖与呼吸综合征病毒宁夏分离株ORF5基因的克隆表达及其表达产物的活性分析  

Cloning and expression of ORF5 gene of porcine reproductive and respiratory syndrome virus NX/HY strain and analysis of activity of the expressed product

作　　者：陈妍[1] 田宏[1] 尹双辉[1] 尚佑军[1] 孙靖[1,2] 刘湘涛[1] 

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农业部畜禽病毒学重点开放实验室,甘肃兰州730046 [2]吉林大学,吉林长春130062

出　　处：《中国兽医科学》2009年第9期774-778,共5页Chinese Veterinary Science

基　　金：国家"十一五"高技术研究发展计划(863)项目(2006AA10A204)

摘　　要：参考已发表的猪生殖与呼吸综合征病毒(PRRSV)ORF5基因序列,设计合成了1对覆盖完整ORF5基因区段的引物,克隆了PRRSV NX/HY株的ORF5基因(登录号为EU600287),并进行了序列比较。将该基因克隆到原核表达载体pGEX-6P-1中,构建了融合表达质粒pGEX-6P-1-5并转化BL21,对表达蛋白进行Western-blot分析。结果表明,NX/HY分离株ORF5基因与PRRSV JX1、CH-1a株的核苷酸序列同源性分别为99.2%和95.2%,推导的氨基酸序列的同源性分别为98.5%和92.5%;层析扫描分析显示,目的蛋白的含量约占菌体总蛋白的30%,且具有良好的反应原性。GP5蛋白的表达为建立相应的血清学诊断方法奠定了基础。According to the ORF5 gene sequence（GenBank accession No. EU200961） of porcine reproduct ive and respiratory syndrome virus（PRRSV） JX-3 strain, a pair of primers was designed and synthesized. The complete ORF5 gene of PRRSV NX/HY strain was amplified, cloned into pMD18-T vector, sequenced and compared with those of other PRRSV strains. Then, the ORF5 gene was subcloned into the expression vector pGEX-6P-1 to construct expression plasmid pGEX-6P-1-5. The pGEX-6P-1-5 was transformed into Escherichia coli BL21（DE3） and induced by IPTG. Sequencing analysis results showed that the ORF5 gene shared 99.2% and 95.2% identity with that of PRRSV JX1 and CH-1a strains respectively,and the deduced amino acid sequence from the ORF5 gene shared 98. 5% and 92. 5% similarity with that of PRRSV JX1 and CH-1a strains respectively. SDS-PAGE and Western-blot analyses showed that the GP5 protein was expressed about 30% in total proteins and had good reactionogenicity. The results provided a basis for establishment of serological method for diagnosis of PRRS.

关 键 词：猪生殖与呼吸综合征病毒 GP5蛋白 原核表达 

分 类 号：S852.659.6[农业科学—基础兽医学]