猪繁殖与呼吸综合征病毒HB-3(cz)株ORF5基因序列分析及原核表达  

Preliminary studies of prokaryotic expression of ORF5 gene of porcine reproductive and respiratory syndrome virus HB-3(cz) strain

作　　者：刘涛[1,2] 陈赛娟[2] 曹洪战[2] 刘子伟[3] 朱子杰[2] 苏霞[2] 孙继国[2] 

机构地区：[1]商务部流通产业促进中心,北京丰台100070 [2]河北农业大学动物科技学院,河北保定071001 [3]河北省保定市北市区动物防疫监督站,河北保定071000

出　　处：《中国兽医杂志》2008年第12期88-91,共4页Chinese Journal of Veterinary Medicine

基　　金：河北省重大技术创新项目(07221008Z)

摘　　要：从河北沧州分离到1株疑似猪繁殖与呼吸综合征病毒,接种Marc-145细胞,经过4代盲传后,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 CH-1a株ORF5基因的核苷酸序列,设计合成一对特异性引物(P1/P2),用RT-PCR方法扩增完整ORF5基因,将扩增产物连接到pGM-T载体并转化克隆菌,阳性重组质粒进行序列测定与分析。再没计另一对引物(P3/P4)从重组载体pGM-ORF5中扩增出缺失了N端28个氨基酸残基的tGP5的编码序列tORF5,克隆入原核表达载体pGEX-6P-1,并成功获得表达,经Western-blotting分析表明,表达蛋白与阳性血清发生特异性反应。为研制PRRSV新型疫苗及诊断试剂奠定了基础。The full-length of ORF5 gene encoding the envelope glycoprotein （GP5） of PRRSV HB-3 （cz） strain was amplified by RT-PCR with a pair of primers （p1/P2） and cloned into plasmid pGM-T. The recombinant plasmid designated as pGM-E was subjected to sequencing and analysis and used as the template to amplify the truncated ORF5 （tORF5） ,which is coding the GP5 lacking the first 28 residues at the amino terminal region ,by the use of primers （p3/ p4）. And the gene was subcloned into prokaryotic expression vector pGEX-6P-1. The recombinant fusion proteins pGEX-tORF5 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 ORF5基因 原核表达 

分 类 号：S852.65[农业科学—基础兽医学]