猪繁殖与呼吸综合征病毒GP5蛋白的原核表达及鉴定

Prokaryotic Expression and Identification of Porcine Reproductive and Respiratory Syndrome Virus GP5 Protein

关键词:  猪繁殖与呼吸综合征病毒  GP5蛋白  原核表达  

Key words:  PRRSV  GP5 protein  prokaryotic expression

摘要:

为获得猪繁殖与呼吸综合征病毒（PRRSV）GP5蛋白，本研究以一株PRRSV NADC30-like毒株为模板，利用RT-PCR扩增出ORF5基因后，将该基因克隆至原核表达载体pCold-TF中，构建了重组质粒pCold-TF-GP5，转化Rosetta （DE3）后用IPTG诱导，经SDS-PAGE、Western blot鉴定，重组蛋白获得了可溶性表达，大小约为72ku。将该蛋白过镍柱纯化后获得浓度为0.5mg/ml的纯化蛋白，为下一步GP5蛋白单克隆抗体的制备奠定物质基础。

Abstract:

In order to obtain the GP5 protein of porcine reproductive and respiratory syndrome virus (PRRSV), the ORF5 gene was amplified by RT-PCR using the PRRSV NADC30-like strain genomic RNA as the template. The ORF5 gene was cloned into the prokaryotic expression vector pCold-TF. The recombinant plasmid pCold-TF-GP5 was constructed and transformed into Rosetta（DE3）and induced by IPTG. The SDS-PAGE and Western blot identification indicates the recombinant protein was soluble and its size was about 72 ku. The protein was purified by nickel column with a concentration of 0.5mg/mL, which laid the material foundation for the preparation of GP5 monoclonal antibody in the next step.