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长白猪γ干扰素基因的原核表达与分析

赵英杰1，3，徐宏军1，李尚波1，王文成1，卫广森2，宣华3

(1. 辽宁益康生物制品有限公司 研发中心，辽宁 辽阳111000； 2. 中国农业科学院 
兰州兽医研究所，甘肃 兰州730046； 3.吉林大学 农学部 畜牧兽医学院，吉林 长春130062）

摘要：  
以含长白猪IFNγ基因的质粒载体为模板，用原核表达引物扩增了长白猪IFNγ基因，将其定向克隆至原核表达载体pET28b的EcoRⅠ和XhoⅠ位点，构建了长白猪IFNγ基因的原核表达载体pET28bPoIFNγ。经酶切、PCR鉴定，表明所构建的重组质粒为特异性长白猪IFNγ基因原核表达质粒。将该重组质粒转化大肠埃希氏菌BL21（DE3）细胞，阳性菌落筛选、SDSPAGE分析以及Westernblotting分析表明，成功构建了表达重组猪IFNγ的大肠埃希氏菌基因工程菌株。亚细胞定位分析表明，目的蛋白经原核表达后主要以不溶性包涵体形式存在，其他少量可溶性目的蛋白存在于细胞浆中。
关键词：  
长白猪； IFNγ； 原核表达； 亚细胞定位； Westernblotting分析
中图分类号：S 859.79: Q 786  文献标识码： A  文章编号：1673-4696(2006)01-0046-06

Prokaryotic expression and analysis of Changbai porcine IFNγ gene

ZHAO Yingjie1,3, XU Hongjun1, LI Shangbo1, WANG Wencheng1, 
WEI Guangsen2, XUAN Hua3

(1. Center for Research and Development, Liaoning Yikang Bioproduct Co., Liaoyang 111000,China;
2. Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046,China；
3. College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062,China)
Abstract: The PoIFNγ gene was inserted into EcoRⅠand XhoⅠmultiple cloning sites of the prokaryotic expression vector pET28b using the expression primers designed according to the sequence of PoIFNγ gene. The pET28bPoIFNγ was identified and analyzed by enzymatic digestion and PCR amplification, and was confirmed to be the IFNγ gene of Changbai porcine. The purified pET28bPoIFNγ was transformed into E.coli BL21(DE3) and the recombinant engineering strain was harvested. Identification by positive clone selection, SDSPAGE and Westernblotting analyses showed that the PoIFNγ gene was recombinated correctly with pET28b in BL21(DE3) cells. Results confirmed that construction of the recombinant engineering strain of BL21(DE3)/ pET28bPoIFNγ was successful. The localization analysis revealed that the rPoIFNγ was mainly present in the cells in the form of inclusion bodies and a very small portion was soluble in cytoplasm.

Key words: Changbai porcine； IFNγ； prokaryotic expression； localization analysis； Westernblotting

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