高致病性猪蓝耳病病毒GST-Nsp2融合蛋白的原核表达  

Prokaryotic Expression of GST-Nsp2 Fusion Protein of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

作　　者：刘金玲[1] 魏澍[2] 潘树德[1] 

机构地区：[1]沈阳农业大学畜牧兽医学院,辽宁沈阳110866 [2]辽宁省动物疫病预防控制中心,辽宁沈阳110164

出　　处：《江西农业大学学报》2012年第3期546-549,共4页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家科技攻关项目(413010209);沈阳农业大学校青年基金项目(200705)

摘　　要：通过对高致病性猪繁殖与呼吸综合征病毒(HPPRRSV)Nsp2基因进行克隆表达以获得蛋白,为该病的诊断预防奠定基础。采用自行设计的引物对高致病性猪繁殖与呼吸综合征病毒进行RT-PCR扩增,将产物基因回收纯化测序后构建PGEX-6P-1-Nsp2原核表达载体,用BL21(DE3)细胞进行转化,挑选阳性菌,用IPTG诱导表达,采用SDS-PAGE电泳和Western-Blot对表达产物进行鉴定。获得了HPPRRSV Nsp2基因序列,成功构建了PGEX-6P-1-Nsp2原核表达载体,经37℃,4 h获得表达产物,将表达产物进行SDS-PAGE和Western-Blot分析,获得了一条约42 ku浓缩蛋白带。本实验的克隆表达产物为高致病性蓝耳病病毒Nsp2目的蛋白。The study was to lay the foundation for the disease prevention and diagnosis by cloning and expressing the Nsp2 gene of highly pathogenic porcine reproductive and respiratory syndrome virus （HPPRRSV）.The primer designed by the authors was used to amplify the Nsp2 gene of Highly Pathogenic Porcine repro respiratory syndrome virus by reverse transcriptase polymerase chain reaction, and the gene was cloned into PGEX-6P-1 vector after the Nsp2 gene of HPPRRSV was purified and sequenced, after that, the recombinant plasmid PGEX-6P-1-Nsp2 was obtained.The full plasmid was transferred into E.coli BL21 （DE3） bacteria and the bacteria containing the positive plasmid were selected, the total expressed protein was harvested and extracted and then identified by SDS-PAGE and Western-Blot. Nsp2 gene of HPPRRSV and the recombinant plasmid PGEX-6P-1-Nsp2 were obtained, and then, the expressed proteins induced with IPTG for 4 hours later at 37 ~C were harvested. The analysis showed that the molecular weight of the fusion protein was approximately 42 ku in agreement with the expect.The cloning and expression product in this study was the Nsp2 protein of HP PRRSV.

关 键 词：高致病性 猪繁殖与呼吸综合征病毒 NSP2基因 原核表达 

分 类 号：S852.651[农业科学—基础兽医学]