人造血相关PBX相互作用蛋白的原核表达及纯化鉴定  

Prokaryotic Expression,Purification and Characterization of the Recombinant Human Hematopoietic PBX-Interacting Protein

作　　者：纪贝贝 赵晖[1] 史平安[2] 徐小洁[2] 黄蓉[1] 李玲[2] 范忠义[2] 郭靖[2] 梁迎春[2] 吕朝辉[2] 叶棋浓[2] 杜楠[1] 

机构地区：[1]解放军总医院第一附属医院,北京100048 [2]军事医学科学院生物工程研究所,北京100850

出　　处：《生物技术通讯》2015年第3期338-341,共4页Letters in Biotechnology

基　　金：国家自然科学基金(31100604;81472589);北京市科技新星计划(Z141102001814055);北京市自然科学基金(7132155);军事医学科学院创新基金转化医学项目(ZHYX003)

摘　　要：目的:构建带His标签的人造血相关PBX相互作用蛋白(HPIP)的原核表达载体,获得His-HPIP融合蛋白,并对其生物学功能进行初步检测。方法:以本实验室保存的pcDNA3.0-HPIP质粒为模板,采用PCR技术扩增HPIP编码序列,将其插入载体p ET-28a(+)中,经Bam HⅠ和HindⅢ双酶切鉴定后转化大肠杆菌Rossate株进行小量诱导,挑选能诱导出His-HPIP的菌液进行融合蛋白的纯化,采用SDS-PAGE和Western印迹检测融合蛋白的纯化效果,采用GST pull-down技术对蛋白的生物学功能进行初步鉴定。结果:双酶切和测序结果表明His-HPIP原核表达质粒构建成功;His pull-down实验证实His-HPIP蛋白和雌激素受体α存在相互作用,说明生物学活性良好。结论:原核表达并纯化出His-HPIP融合蛋白,为进一步研究HPIP在肿瘤发生发展中的功能奠定了基础。Objective： To construct the prokaryotic expression vector of human hematopoietic PBX-interacting pro-tein（HPIP） with His-tag and detect the activity of recombinant fusion protein.Methods： Human HPIP gene wasobtained from pcDNA3.0-HPIP plasmid by PCR and cloned into the prokaryotic expression vector p ET- 28a（＋）.The correct recombinant plasmid was introduced into E.coli Rossate.The expressed recombinant protein was puri-fied by Ni-NTA beads and identified by SDS-PAGE and Western blot analysis.The function of purified His-HPIP protein was detected by GST pull-down assay.Results： HPIP prokaryotic expression vector labeled with His-tag was successfully constructed by double digestion identification.The inserted fragment was confirmed correct bysequencing.His pull-down assay showed that His-HPIP could interact with estrogen receptor-α in vitro,confirm-ing its known function.Conclusion： The prokaryotic expression vector of His- HPIP was obtained successfully,which laid foundation for the further study of the role of HPIP in cancer development and progression.

关 键 词：人造血相关PBX相互作用蛋白 原核表达 纯化 

分 类 号：Q78[生物学—分子生物学]