中文摘要：

目的：构建基于转录激活因子（TAT）技术的细胞外信号调节激酶（ERK）2及其无活性突变体ERK2（AF）原核载体并在大肠杆菌BL21（DE3）中表达。方法：采用聚合酶链式反应（PCR）技术从pcDNA3-Flag-ERK2和pcDNA3-Flag-ERK2（AF）质粒中扩增出ERK2和ERK2（AF）基因，插入pET14b-His-TAT载体中，构建重组质粒pET14b-His-TAT-ERK2和pET14b-His-TAT-ERK2（AF），并诱导原核表达、纯化融合蛋白。采用Western blot方法检测表达蛋白的His标签。结果：酶切和测序结果表明，扩增的ERK2和ERK2（AF）基因正确，大小为1082bp；10%SDS-PAGE凝胶电泳可见原核表达纯化得到高纯度目的蛋白，大小为41 kD；Western blot检测显示，原核蛋白带有His标签。结论：成功构建了ERK2和ERK2（AF）的原核表达载体，该载体能在大肠杆菌中表达，纯化得到了His-TAT-ERK2和His-TAT-ERK2（AF）蛋白，为研究ERK2蛋白的功能提供了重要工具。

英文摘要：

Objective： To construct pET14b-His-TAT-extracellular-signal regulated protein （ERK）2 and pET14b-His-TAT-ERK2 （AF）-incompentence mutant prokaryotic expression vectors based on the trans-acting activator of transcription （TAT） transduction technique, and express in Bacillus coil BL21 （DE3）. Methods： The ERK2 and ERK （AF） gene were amplified from pcDNA3-Flag-ERK2 and pcDNA3-Flag-ERK2 （A.F-） plasmids by polymerase chain reaction （PCR） and cloned into pET14b-His-TAT vector. The resulting plasmids were verified by enzyme digestion and DNA sequencing. The ERK2 and ERK2 （AF） proteins were expressed in BL21 （DE3）, and Western blot was used to detect their His tag. Results： The result of DNA sequencing and enzyme digestion showed that the cloned ERK2 and ERK2 （AF） genes were correct to be 1082 bp. HT-ERK2 and HT-ERK2 （AF） protein were expressed in BL21 （DE3） ,being approximately 41 kD. His tag in two proteins was verified by West- ern blot. Conclusion： pHis-TAT-ERK2 and pHis-TAT-ERK2 （AF）-incompentence mutant prokaryotic expression vectors are constructed successfully. HT-ERK2 and HT-ERK2 （AF） proteins are obtained, and they might serve as important tools to study the protein function of ERK2.