摘要
为了获得具有抗病毒活性的水牛γ干扰素(interferon-γ,IFN-γ)蛋白,试验采用PCR方法扩增水牛IFN-γ基因编码区序列,将其与pET-32a载体连接,构建重组表达质粒pET32a-IFN-γ,并将其转化至大肠杆菌BL21感受态细胞中,用IPTG诱导表达目的蛋白,对表达的重组蛋白进行SDS-PAGE分析、纯化、Western-blot鉴定与质谱分析,以及抗病毒活性检测。结果表明:水牛IFN-γ基因编码区序列PCR扩增产物大小约为520 bp;重组表达质粒pET32a-IFN-γ的双酶切和测序鉴定结果均正确;阳性菌株经诱导后表达的重组蛋白大小约为39 ku,主要以可溶性蛋白形式表达,且能与His标记小鼠源单克隆抗体发生特异性反应,其肽段氨基酸序列与目的蛋白氨基酸序列完全匹配;纯化后的重组蛋白IFN-γ能够有效抑制牛病毒性腹泻病毒引起的细胞病变,抗病毒活性约为294.1 U/mg。说明水牛IFN-γ蛋白在大肠杆菌中成功表达,并具有一定抗病毒活性。
In order to obtain interferon-γ(IFN-γ) protein with antiviral activity, in this study, the coding region of buffalo IFN-γ gene was amplified by PCR, and the recombinant plasmid pET32 a-IFN-γ was constructed by linking it with pET-32 a vector. The recombinant plasmid pET32 a-IFN-γ was transformed into E.coli BL21 receptor cells, and the target protein was expressed by IPTG induction. SDS-PAGE analysis, purification, Western-blot identification, mass spectrometry analysis and antiviral activity detection were performed on the recombinant protein. The results showed that the PCR product size of buffalo IFN-γ coding region was about 520 bp. The recombinant plasmid pET32 a-IFN-γ was identified by double digestion and sequencing. The recombinant IFN-γ of the positive strain was about 39 ku in size, mainly expressed in soluble form, and could react specifically with His labeled murine monoclonal antibody. The amino acid sequence of the peptide was exactly matched with that of the target protein. The purified recombinant IFN-γ could effectively inhibit the cytopathic effect caused by bovine viral diarrhea virus on bovine kidney cells, and the antiviral activity was about 294.1 U/mg.These results indicated that buffalo IFN-γ protein was successfully expressed in E.coli and had certain antiviral activity.
作者
李小凤
谢芝勋
武晓倩
范晴
谢志勤
韦悠
谢丽基
万丽军
王盛
张艳芳
黄娇玲
罗思思
LI Xiaofeng;XIE Zhixun;WU Xiaoqian;FAN Qing;XIE Zhiqin;WEI You;XIE Liji;WAN Lijun;WANG Sheng;ZHANG Yanfang;HUANG Jiaoling;LUO Sisi(Guangxi Veterinary Research Institute,Nanning 530001,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2022年第14期124-128,140,共6页
Heilongjiang Animal Science And veterinary Medicine
基金
广西科技重大专项(桂科AA17204057)
广西八桂学者专项(2019A50)
国家万人计划领军人才专项(W02060083)。
关键词
水牛
Γ干扰素
原核表达
抗病毒活性
质谱分析
buffaloes
IFN-γ
prokaryotic expression
antiviral activity
mass spectrometry
