牛eIF5基因原核表达与蛋白纯化  

Prokaryotic expression and protein purification of gene bovine eIF5

作　　者：高学军[1] 臧艳丽[1] 魏诚杰 骆超超[1] 

机构地区：[1]东北农业大学黑龙江省高校农业生物功能基因重点实验室,哈尔滨150030

出　　处：《东北农业大学学报》2016年第6期68-73,共6页Journal of Northeast Agricultural University

基　　金：国家高技术研究发展计划(863)项目(2013AA102504-03)

摘　　要：研究应用PCR方法扩增牛e IF5基因编码序列,通过Eco RⅠ和SalⅠ酶切位点将其定向插入p GEX-4T-1载体,构建原核表达重组质粒p GEX-4T-1-e IF5,并转化大肠埃希菌BL21。酶切和测序鉴定正确后,用异丙基硫代-β-D-半乳糖苷(IPTG)大量诱导表达,利用GST-Sepharose 4B亲和珠纯化,SDS-PAGE和Western Blot鉴定。结果表明,原核表达载体p GEX-4T-1-e IF5构建成功,Western Blot证实融合蛋白大量表达,纯化后获得GSTe IF5融合蛋白,为深入研究牛e IF5蛋白功能奠定基础。The bovine eIF5 gene coding sequence was amplified by polymerase chain reaction （PCR） and was cloned into Eco R I and SalⅠ sites of pGEX-4T-1 vector to construct the recombinant plasmid pGEX-4T-1-eIF5. The recombinant plasmid was identified by restriction enzyme digestion and sequencing. Then it was transformed into BL21, induced by IPTG and purified by GST-Sepharose 4B beads, identified by SDS-PAGE and Western Blot analysis. Results showed that the prokaryotic expression vector pGEX-4T-1-eIF5 was successful y constructed. Western Blot confirmed the recombinant protein GST-eIF5 were expressed and purified. This study provided the basis for the further research on the protein function of bovine eIF5.

关 键 词：牛 eIF5 克隆 原核表达 纯化 

分 类 号：S823[农业科学—畜牧学] Q785[农业科学—畜牧兽医]