猪干扰素-γ基因的表达、纯化及活性分析  

Expression,purification and bioactivity analysis of porcine interferon-gamma gene

作　　者：潘晓梅[1,2] 窦永喜[1] 骆学农[1] 刘琼[1,2] 张冬峰[1,2] 岳城[2] 才学鹏[1] 

机构地区：[1]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,甘肃省动物寄生虫病重点实验室,甘肃兰州730046 [2]新疆农业大学动物医学院,新疆乌鲁木齐830052

出　　处：《甘肃农业大学学报》2009年第2期7-12,25,共7页Journal of Gansu Agricultural University

基　　金：国家高科技研究发展计划“863”项目(2006AA10A207);甘肃省重大科技专项(ZGS063-A43-013)

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.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 SKI. 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 × 10^3 U · mg^-1 , the activity of IFN-γ against FMDV serotype O was about 2.56× 10^5 U · mg^-1.

关 键 词：猪IFN-γ 表达 蛋白纯化 活性分析 

分 类 号：S852.4[农业科学—基础兽医学]