猪繁殖与呼吸综合征病毒河北地方株ORF6基因的克隆及原核表达  

Preliminary studies of prokaryotic expression of ORF6 gene of porcine reproductive and respiratory syndrome virus HB-3(cz) strain

作　　者：王娇[1] 孙继国[1] 陈赛娟[1] 刘涛 王坤[1] 赵泽坤[1] 

机构地区：[1]河北农业大学动物科技学院,河北保定071001 [2]商务部流通产业促进中心,北京100070

出　　处：《中国动物检疫》2009年第4期34-36,共3页China Animal Health Inspection

基　　金：河北省重大技术创新项目资助(07221008Z)

摘　　要：从河北沧州分离到一株疑似猪繁殖与呼吸综合征病毒,接种Marc-145细胞,经4代盲传,出现细胞病变,经鉴定为PRRSV,命名为HB-3(cz)株。根据GenBank公布的PRRSV JXA1株ORF6基因的核苷酸序列,设计并合成一对特异性引物(P1/P2),用RT-PCR方法扩增完整ORF6基因,将扩增产物连接到pGM-T载体并转化克隆菌,阳性重组质粒PGM-M进行序列测定与分析。后将克隆质粒PGM-M双酶切后连接原核表达载体pET-32a(+),在Rosseta-DE3中成功获得表达,经Western-blotting分析表明,表达蛋白与阳性血清发生特异性反应。The full-length of ORF6 gene encoding the envelope glycoprotein （GP6） of PRRSV HB-3（cz） strain isolated from Cangzhou HeBei was amplified by RT-PCR with a pair of specific primers （p1/ p2） designed according to the relevant sequence of PRRSV JXA1 strain, and then cloned into plasmid pGM-T,the recombinant plasmid designated as pGM-M was subjected to sequencing and analysis. After identification by restriction endonclease digestion,the gene was subcloned into prokaryotic expression vector pET-32 a （＋）. The recombinant fusion proteins pET-M were highly expressed in Escherichia coli BL21. Western-blotting showed that the recombinant protein could react with the porcine polyclonal antibody against PRRSV.

关 键 词：猪繁殖与呼吸综合征病毒 河北分离株 ORF6基因 原核表达 

分 类 号：S852.659.6[农业科学—基础兽医学]