重组猪干扰素α发酵纯化与抗病毒活性测定
Expression Purificationand Bioactivity of Recombinant Porcine Interferon-α(IFNα)
陈红艳 1施忠芬 1胡媛媛 1法林荣 1张涛2
作者信息
- 1. 云南农业职业技术学院,云南昆明 650000
- 2. 云南省昆明市晋宁区农业农村局,云南昆明 650000
- 折叠
摘要
本研究根据大肠杆菌的密码子偏好性,对UniProt数据库中的猪干扰素α序列(编号:P49879)进行优化,合成了相应的基因片段,并构建了 pET-28a-INF-α原核表达载体.将重组载体转化到大肠杆菌BL21(DE3)中,用IPTG诱导表达,经过20 L发酵罐培养24 h,在OD600为108时收获细胞.表达的猪干扰素α主要以包涵体的形式存在,经过变性、复性、镍亲和纯化与酶切等步骤,得到了无标签的猪干扰素α蛋白.SDS-PAGE电泳显示,该蛋白的分子量约为19 kDa,纯度高达95％.细胞病变抑制法测定表明,该蛋白的抗病毒活性为1.28× 107 IU/mg.本研究成功获得了高纯度、高活性的猪干扰素α蛋白,为进一步推进蛋白药物在临床上的应用奠定了基础,为进一步重组蛋白工业化生产提供了理论依据.
Abstract
In this study,the sequence of porcine interferon α(No.P49879)in the UniProt database was op-timized according to the codon preference of Escherichia coli,the corresponding gene fragment was synthesized,and the pET-28a-INF-α prokaryotic expression vector was constructed.The recombinant vector was trans-formed into Escherichia coli BL21(DE3),and expression was induced by IPTG.After incubation in a 20 L fer-menter for 24 h,the cells were harvested with an OD600of 108.The expressed porcine interferon α mainly existed in the form of inclusion bodies,and the label-free porcine interferon α protein was obtained through the steps of de-naturation,repletion,nickel-column purification,and enzymatic digestion.SDS-PAGE electrophoresis showed that the The molecular weight of the protein was about 19 kD and the purity was up to 95％.The cytopathic inhibi-tion assay showed that the antiviral activity of the protein was 1.28 x 107 IU/mg.In this study,the high purity and high activity of porcine interferon α protein was successfully obtained,which lays the foundation for further advan-cing the clinical application of the protein drug,and provides the basis for the commercial production.
关键词
猪干扰素α/表达/发酵/纯化/抗病毒活性Key words
Porcine interferon alpha/Expression/Fermentation/Purification/Antiviral activity引用本文复制引用
基金项目
云南省教育厅科学研究基金(2020J1322)
出版年
2024
NETL
NSTL
万方数据