新的锌指蛋白Mip1的DNA结合序列的筛选  

Screening of DNA blinding site of a novel zinc finger protein Mip1

作　　者：蒋磊[1] 王慷慨[1] 陈广文[1] 刘可[1] 邓恭华[1] 涂自智[1] 刘梅冬[1] 王桂良[1] 肖献忠[1] 

机构地区：[1]中南大学湘雅医学院病理生理学教研室,湖南长沙410078

出　　处：《中国现代医学杂志》2008年第8期1048-1051,共4页China Journal of Modern Medicine

基　　金：国家重点基础研究发展规划项目(973)(G2000056908);国家自然科学基金资助项目(30170373;30300138)

摘　　要：目的筛选一个新的锌指蛋白Mip1的DNA结合位点。方法将Mip1cDNA全长克隆入原核表达质粒pGEX-4T-1构建了Mip1的原核表达质粒pGEX-Mip1。用谷胱甘肽亲和层析纯化GST-Mip1融合蛋白。通过固定化的GST-Mip1,从寡核苷酸文库中俘获Mip1的结合的寡核苷酸片段;将所得寡核苷酸片段插入T-载体,通过测序、序列比对得到DNA结合位点。结果锌指蛋白Mip1的融合蛋白能在大肠杆菌中高效表达,能与固定化还原性谷胱甘肽很好地亲和而得到纯化;用固定化的Mip1融合蛋白俘获到了其结合的寡核苷酸序列,通过测序、序列比对,得到了Mip1的DNA结合位点。结论Mip1的DNA结合位点的核心序列为G/TCCTA,Mip1的DNA结合位点的成功确定为Mip1功能的深入研究打下了基础。[Objective] To screen the DNA binding site of a novel zinc finger protein named Mipl. [Methods] Mipl full-length ORF was cloned into a bacterial expression vector pGEX-4T-1 to construct pGEX-Mipl, pGEX- Mipl was overexpressed in E.eoli M15 to generate soluble GST-Mipl fusion protein, and the GST-Mipl was purified by glutathione affinity chromatography and immobilized on Sepharose beads to capture putative target DNA binding site from the oligonucleotide library. The selected oligonueleotides were gel-purified and inserted into T vector for sequencing and the consensus sequence was derived by sequence alignment. [Results] GST-Mipl fusion protein was over-expressed in E.coli M15 and purified by affinity chromatography. Using immobilized-Mipl beads, a lot of oligonueleotides were fished out from the oligonueleotides library. A consensus sequence which may be the DNA binding site of Mipl, was derived from sequence alignment. [Conclusion] The DNA binding site of Mipl may be the consensus sequence G/TCCTA.

关 键 词：新基因 Mipl 锌指蛋白 表达与纯化 寡核苷酸文库 DNA结合位点 

分 类 号：R36[医药卫生—病理学]