小鼠干扰素应答基因(Ifrg15)的克隆、原核表达及其融合蛋白的亲和纯化  

Cloning and Prokaryotic Expression of Mouse Interferon Responsive Gene (Ifrg15) and Affinity Purification of Its Fusion Protein

作　　者：丁彪[1,2] 刘一飞[1] 张运海[2] 李文雍[1] 

机构地区：[1]阜阳师范学院生命科学学院胚胎发育与生殖调节安徽省重点实验室,阜阳236041 [2]安徽农业大学动物科技学院,合肥230036

出　　处：《农业生物技术学报》2010年第6期1168-1172,共5页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No30871415;31071310);安徽高校省级自然科学研究规划重点项目(NoKJ2008A136)共同资助

摘　　要：为研究一个可能的干扰素应答基因(interferon responsive gene15,Ifrg15)所编码蛋白质的生物学功能,对昆明小鼠(Mus musculus)基因组DNA进行PCR扩增获得Ifrg15(393bp),并将其克隆到pGEM-T载体,经测序鉴定后用EcoRⅠ和XhoⅠ酶切下Ifrg15基因片段并和双酶切后的表达载体pET-41(c)进行连接,然后转化入大肠杆菌(Escherichia coli)BL21(DE3)感受态细胞。对重组克隆用IPTG诱导融合蛋白的表达,并进行分离纯化。结果表明,本实验在E.coliBL21(DE3)菌株中成功地高效表达、并在变性亲和层析条件下成功纯化了GST&6xHis-Ifrg15融合蛋白。In order to study the function of a putative interferon responsive gene （Ifrg15）, the genomic DNA of Kunming White mouse（Mus musculus） was purified and used as the PCR template for amplifying the Ifrg15 coding region. The PCR product was cloned into pGEM-T vector and confirmed by DNA sequencing. The Ifrg15 fragment was subcloned into pET-41（c） vector by EcoR Ⅰ and Xho Ⅰ double-digestion. Escherichia coli BL21（DE3） transformants were then selected and tested by IPTG induction for high level expression of GST6xHis-Ifrg15 fusion protein. The results showed that mouse Ifrg15 gene was successfully cloned and expressed in the prokaryotic expression system. By using denaturing condition in the affinity chromatographic step, a decent level of purification of GST6xHis-Ifrg15 fusion protein was achieved.

关 键 词：干扰素应答基因(Ifrg15) 基因克隆 原核表达 亲和层析 

分 类 号：Q550.3[生物学—生物化学]