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)

作　　者：陈红艳[1] 施忠芬[1] 胡媛媛[1] 法林荣[1] 张涛 CHEN Hongyan;SHI Zhongfen;HU Yuanyuan;FA Linrong;ZHANG Tao(Yunnan vocational college of agricultural,Kunming 650000,China;Jinning County Agriculture and rural bureau,Kunming 650000,China)

机构地区：[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

出　　处：《青海畜牧兽医杂志》2024年第3期10-15,共6页Chinese Qinghai Journal of Animal and Veterinary Sciences

基　　金：云南省教育厅科学研究基金项目资金资助(2020J1322)。

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OD 600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1.28×10^(7 )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。In this study,the sequence of porcine interferonα(No.P49879)in the UniProt database was optimized according to the codon preference of Escherichia coli,the corresponding gene fragment was synthesized,and the pET-28a-INF-αprokaryotic expression vector was constructed.The recombinant vector was transformed into Escherichia coli BL21(DE3),and expression was induced by IPTG.After incubation in a 20 L fermenter for 24 h,the cells were harvested with an OD 600 of 108.The expressed porcine interferonαmainly existed in the form of inclusion bodies,and the label-free porcine interferonαprotein was obtained through the steps of denaturation,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 inhibition assay showed that the antiviral activity of the protein was 1.28×10^(7 )IU/mg.In this study,the high purity and high activity of porcine interferonαprotein was successfully obtained,which lays the foundation for further advancing 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 

分 类 号：S852.65[农业科学—基础兽医学]