PHD2基因原核表达载体的构建及其在大肠埃希菌中的表达 |
| |
引用本文: | 杨丰旭,;田晶,;高鹏,;许国旺,;张卓然.PHD2基因原核表达载体的构建及其在大肠埃希菌中的表达[J].中国微生态学杂志,2014(6):627-630. |
| |
作者姓名: | 杨丰旭 ;田晶 ;高鹏 ;许国旺 ;张卓然 |
| |
作者单位: | [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 |
| |
基金项目: | 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。
|
关 键 词: | PHD2 In-Fusion技术 Nus·Tag 大肠埃希菌 |
Construction of a prokaryotic expression vector of PHD2 and its expression in E. coli |
| |
Institution: | YANG Feng-xu , TIAN Jing , GAO Peng, XU Guo-wang , ZHANG Zhuo-ran( 1. School of Bioengineering, Dalian Polytechnic University, Dalian 116034, China; 2. CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; 3. Dalian Society of Microbiology, Dalian 116033, China) |
| |
Abstract: | 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. |
| |
Keywords: | PHD2 In-Fusion technology Nus Tag Escherichia coli |
本文献已被 维普 等数据库收录! |
|