作 者：
潘晓梅;窦永喜;骆学农;刘琼;张冬峰;岳城;才学鹏
单 位：
新疆农业大学动物医学院;中国农业科学院兰州兽医研究所
摘 要：
依照猪IFN-γ基因序列设计引物,将该基因克隆至原核表达载体pET-30a,构建pET-30a-pIFN-γ重组表达载体,转化入寄主菌BL21(DE3),经PCR、双酶切、测序鉴定正确后,用IPTG进行诱导表达,可溶性的表达产物分别经镍柱和Sephadex-G100纯化;包涵体经DOC洗涤、SKL变性溶解、透析复性进行纯化,然后进行SDS-PAGE、Western-blot分析,并用细胞病变抑制法进行重组猪IFN-γ活性测定.结果表明:试验得到了高纯度的重组pIFN-γ,且纯化的重组猪IFN-γ蛋白具有较高的干扰病毒复制的活性,对PRV的活性为2.0×103U.mg-1,而对标准O型口蹄疫病毒的活性为2.56×105U.mg-1.
译 名：
Expression,purification and bioactivity analysis of porcine interferon-gamma gene
作 者：
PAN Xiao-mei1,2,DOU Yong-xi1,LUO Xue-nong1,LIU Qiong1,2,ZHANG Dong-feng1,2,YUE Cheng2,CAI Xue-peng1(1.Key Laboratory of Veterinary Parasitology of Gansu Province,State Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;2.College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China)
关键词：
pIFN-γ;expression;protein purification;bioactivity analysis
摘 要：
A pair of primer were designed according to the sequence of porcine IFN-γ,then the gene was cloned into pET-30 a prokaryotic expression vector,recombinant expression vector pET-30 a-pIFN-γ was constructed.The recombinant plasmid was transformed into E.coli BL21(DE3) and then identified by PCR,double enzyme digesting and sequencing,and induced by IPTG.The soluble expressed product was purified through Nickel-affinity chromatography column,the protein was further purified by Sephadex-G100.The inclusion body was washed by DOC,then dissolved with SKL and subsequently renatured by dialysis,and the expressed protein was analyzed by SDS-PAGE and Western-blot.It was confirmed that the highly purified pET-30a-IFN-γ was harvested.Antiviral effect of the purified product was tested by cytopathogenic effect inhibition assay,the results indicated that the purified product had a higher activity of interferencing virus replication,the activity of IFN-γ against PRV was about 2.0×103 U·mg-1,the activity of IFN-γ against FMDV serotype O was about 2.56×105 U·mg-1.
