PHD2基因原核表达载体的构建及其在大肠埃希菌中的表达  

Construction of a prokaryotic expression vector of PHD2 and its expression in E. coli

作　　者：杨丰旭[1,2] 田晶[1] 高鹏[2] 许国旺[2] 张卓然 

机构地区：[1]大连工业大学生物工程学院,辽宁大连116034 [2]中国科学院大连化学物理研究所分离分析化学重点实验室,辽宁大连116023 [3]大连市微生物学会,辽宁大连116033

出　　处：《中国微生态学杂志》2014年第6期627-630,共4页Chinese Journal of Microecology

基　　金：辽宁省自然科学基金项目(2013020167);国家自然科学基金项目(81372695)

摘　　要：目的构建PHD2基因原核表达载体pET-43.1b(+)-PHD2,实现Nus-PHD2融合蛋白在大肠埃希菌中的可溶性表达。方法用SacⅠ酶切pET-43.1b(+)制备线性化载体,设计与线性化载体两端具有至少15个同源序列的特异性引物,以真核重组质粒pCMV6-Entry-EGLN1为模板,PCR法扩增PHD2目的基因。采用In-Fusion技术构建原核表达载体pET-43.1b(+)-PHD2,并将其导入大肠埃希菌BL21(DE3)中诱导表达。用SDS-PAGE和Western blot分析并鉴定表达出的融合蛋白。用Ni-NTA亲和层析法纯化目的蛋白。结果成功构建了PHD2原核表达载体;SDS-PAGE结果显示融合蛋白以可溶性形式表达;Western blot鉴定表明融合蛋白可以与PHD2单克隆抗体特异性结合。结论实现了Nus-PHD2融合蛋白在大肠埃希菌中的可溶性表达,为PHD2生物学功能的研究奠定了基础。Objective To achieve soluble expression of Nus-PHD2 fusion protein in E. coli. Methods pET-43.1 b （ ＋ ） vector was digested by SacI and the linearized vector was purified. PHD2 gene was amplified from pCMV6- Entry-EGLN1 plasmid. Specific In-Fusion PCR primers were designed to make the PCR products contain ends that were homologous to the linearized vector. PHD2 gcne was cloned into the linearized pET-43, lb（ ＋ ） vector using In-Fusion technology. The recombinant expression plasmid was then transformed into BL21 （DE3） and induced to express the Nus-PHD2 fusion protein. The recombinant protein was analyzed and identified by SDS-PAGE and Western blot. And the target protein was purified by Ni-NTA His · Bind. Results The prokaryotic expression plasmid pET-43.1b（ ＋ ）-PHD2 was successfully constructed. The recombinant protein was solubly expressed and could specifically react with anti-PHD2 antibody. Conclusion Soluble expression of Nus-PHD2 fusion protein was achieved in E. coli, which lay a foundation for further study on the biological functions of PHD2.

关 键 词：PHD2 In-Fusion技术 Nus·Tag 大肠埃希菌 

分 类 号：Q93[生物学—微生物学]