猪生殖与呼吸综合征病毒分离株dNsp2基因的原核表达  

Expression of the deleting-Nsp2 gene of porcine reproductive and respiratory syndrome virus in Escherichia coli

作　　者：梁继望[1,2] 吴发兴[2] 亓传德[2,3] 张志[2] 刘爽[2] 张燕霞[2] 阿合买提[1] 李晓成[2] 

机构地区：[1]新疆农业大学动物医学院,新疆乌鲁木齐830052 [2]中国动物卫生与流行病学中心,山东青岛266032 [3]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2009年第7期622-625,共4页Chinese Veterinary Science

基　　金：国家"十一五"科技支撑计划项目(2007BAD86B05)

摘　　要：参考GenBank中收录的猪生殖与呼吸综合征病毒(PRRSV)JXA1株Nsp2基因序列,设计了1对引物用于扩增截短的Nsp2(deleting Nsp2,dNsp2)基因,用BamHⅠ+XhoⅠ双酶切后将目的基因插入到原核表达载体pET-32a(+)中,转化大肠杆菌BL21(DE3)进行表达,经SDS-PAGE电泳和Western-blot检测,dNsp2基因得到了高效表达,融合蛋白的分子质量约为36.5 ku,表达产物以可溶性方式存在于表达上清液中,表达量可达菌体总蛋白的30%以上,表达产物能被PRRSV变异株抗体所识别,具有较好的生物学活性。PRRSV变异株dNsp2基因的高效表达为该病毒ELISA检测方法的建立及应用奠定了基础。The specific primers were synthesized according to the sequence of Nsp2 gene from porcine reproductive and respiratory syndrome virus published in GenBank. Then the amplified deleting Nsp2 （dNsp2） fragment with Xho Ⅰ and BamH Ⅰ sites was cloned into the vector pET 32a（＋） and the recombinant fusion protein was expressed in Escherichia coli BL21（DE3）. The total mass of the fusion protein with 36.5 ku was confirmed by SDS PAGE and Western blotting. The protein also existed in the supernatant and contained 30 % of the total somatic protein. Western blotting verified that the recombinant protein could be recognized by the porcine variant strain polyclonal antibody against PRRSV, and it had a greater biological activity. The high level expression of dNsp2 gene laid a basis for the establishment of an ELISA for detection of PRRSV.

关 键 词：猪生殖与呼吸综合征病毒 dNsp2基因 原核表达 

分 类 号：S852.659.6[农业科学—基础兽医学]