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.
出处
《动物医学进展》
CSCD
北大核心
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)
关键词
猪繁殖与呼吸综合征病毒
NSP2基因
原核表达
Porcine reproductive and respiratory syndrome virus
Nsp2 gene
prokaryotic expression
