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猪繁殖与呼吸综合征病毒GP5蛋白的原核表达及鉴定
秦义娴,刘丹,高金源,周飞燕,吴华伟
0
(中国兽医药品监察所)
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。
关键词:  猪繁殖与呼吸综合征病毒  GP5蛋白  原核表达  
DOI:
投稿时间:2020-12-13修订日期:2021-02-01
基金项目:中国兽医药品监察所所级课题(201802)
Prokaryotic Expression and Identification of Porcine Reproductive and Respiratory Syndrome Virus GP5 Protein
(China Institute of Veterinary Drug Control)
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.
Key words:  PRRSV  GP5 protein  prokaryotic expression

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