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| 猪干扰素-γ基因的表达、纯化及活性分析 | |
| 引用本文： | 潘晓梅,窦永喜,骆学农,刘琼,张冬峰,岳城,才学鹏. 猪干扰素-γ基因的表达、纯化及活性分析[J]. 甘肃农业大学学报, 2009, 44(2) |
| 作者姓名： | 潘晓梅 窦永喜 骆学农 刘琼 张冬峰 岳城 才学鹏 |
| 作者单位： | 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;新疆农业大学动物医学院,新疆,乌鲁木齐,830052 2. 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; 3. 新疆农业大学动物医学院,新疆,乌鲁木齐,830052 4. 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;重点实验室,甘肃兰州,730046 |
| 基金项目： | 国家高技术研究发展计划(863计划)，甘肃省重大科技专项基金 |
| 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. |
| 关 键 词： | 猪IFN-γ 表达 蛋白纯化 活性分析 |
Expression,purification and bioactivity analysis of porcine interferon-gamma gene |
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| PAN Xiao-mei,DOU Yong-xi,LUO Xue-nong,LIU Qiong,ZHANG Dong-feng,YUE Cheng,CAI Xue-peng. Expression,purification and bioactivity analysis of porcine interferon-gamma gene[J]. Journal of Gansu Agricultural University, 2009, 44(2) | |
| Authors: | PAN Xiao-mei DOU Yong-xi LUO Xue-nong LIU Qiong ZHANG Dong-feng YUE Cheng CAI Xue-peng |
| Affiliation: | PAN Xiao-mei1,2,DOU Yong-xi1,LUO Xue-nong1,LIU Qiong1,ZHANG Dong-feng1,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) |
| Abstract: | 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. |
| Keywords: | pIFN-γ expression protein purification bioactivity analysis |
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