猪繁殖与呼吸综合征病毒TJM株Nsp2基因缺失片段的原核表达及免疫活性分析  

Prokaryotic Expression and Immunogenicity Analysis of Deleted Region of PRRSV TJM Strain Nsp2 Gene

作　　者：黄双[1,2] 王凤雪[2] 温永俊[2] 高维凡[1] 

机构地区：[1]沈阳农业大学,辽宁沈阳110866 [2]中国农业科学院特产研究所吉林省特种经济动物分子生物学国家重点实验室,吉林长春130122

出　　处：《动物医学进展》2014年第3期15-18,共4页Progress In Veterinary Medicine

基　　金：国家863计划项目(2011AA10A213);吉林省特种经济动物疫病防控研究创新团队项目(20121823)

摘　　要：猪繁殖与呼吸综合征病毒(PRRSV)TJ株在细胞克隆纯化后得到非结构蛋白2(Nsp2)缺失的致弱毒株TJM株,根据GenBank数据库中公布的PRRSV TJ株F3代全基因序列设计合成引物,采用RTPCR方法扩增了PRRSV TJM株缺失片段dNsp2,利用原核表达载体pGEX-6p-1构建重组质粒GSTdNsp2,将重组质粒转入大肠埃希菌BL21(DE3)中表达,获得了可溶性表达的融合蛋白(GST-dNsp2)。经亲和柱层析纯化后,得到纯化的GST-dNsp2蛋白,含量1.6mg/mL。用PreScissionTM蛋白酶对融合蛋白GST-dNsp2进行解离,获得浓度为0.41mg/mL的目的蛋白dNsp2。对GST-dNsp2及dNsp2进行Western blot鉴定,证明表达的蛋白具有良好的反应性。本研究获得的GST-dNsp2及dNsp2为ELISA方法鉴别检测PRRSV TJ株与TJM株提供了基础资料。An attenuated porcine reproductive and respiratory syndrome virus （PRRSV）TJM strain with Nsp2 deletions derived from a highly pathogenic PRRSV TJ strain by cloning and purificationon Marc-145 cells.The specific primers were synthesized according to the sequence of porcine reproductive and respirato-ry syndrome virus TJ strain F3 published in GenBank.The deleted region of PRRSV TJM strain Nsp2 was amplified by RT-PCR and cloned into pGEX-6p-1 to construct the recombinant plasmid GST-dNsp2.The recombinant vector was transformed into E.coli BL21（DE3）and expressed.After purification with affinity column,GST-dNsp2 protein was purified and concentration can be up to 1 .6 mg/mL.The targeted protein dNsp2 was dissociated from fusion protein GST-dNsp2 by the PreScissionTM protease,concentration was 0.41 mg/mL.The results showed GST-dNsp2 and dNsp2 had good immunologic activities.The high level expression of dNsp2 laid a basis for establishment of an ELISA for distinguishing PRRSV TJ strain from TJM strain.

关 键 词：猪繁殖与呼吸综合征病毒 NSP2基因 原核表达 

分 类 号：S852.659.6[农业科学—基础兽医学]