人核受体Nurr1基因的克隆和表达及产物纯化(英文)

Molecular Cloning, Expression and Purification of Human Nuclear Receptor Nurr1

核受体相关因子 1(nuclearreceptor relatedfactor 1,Nurr1)是主要表达于中脑黑质及腹侧被盖区多巴胺能神经元的一种转录因子 ,属于核受体超家族成员 ,其功能性配体尚未被确认 .研究表明 ,Nurr1对中脑多巴胺神经元的发育、存活以及成熟后功能的维持具有特殊重要意义 .如能找到它的特异性配体 ,将为最终筛选出治疗帕金森病等中枢多巴胺失调性疾病的药物或化学合成先导物打下基础 .为了获取Nurr1蛋白以标定其配体以及研究蛋白质间的相互作用 ,采用RT PCR技术 ,从人胚中脑组织特异性扩增及克隆了人Nurr1cDNA ,并获得一个在氨基端缺失 35 0bp碱基的Nurr1突变体 .将正常的Nurr1基因片段亚克隆至表达载体pET2 8a ,分别在TNTRT7偶联网织红细胞溶胞系统和大肠杆菌BL2 1(DE3)中获得表达 ,均以可溶性形式存在 ,且产自于体外转录 翻译系统的真核表达Nurr1蛋白已标记上同位素3 5S .Western印迹分析表明 ,所表达的重组目的蛋白具有特异的免疫反应性 .经Ni NTA亲和层析 ,得到了初步纯化的rhNurr1蛋白 .

In order to obtain Nurr1 protein for further ligand screening, protein-protein interaction studying and protein structure analyzing, the full-length fragment of the human Nurr1 coding region from human fetal midbrain tissue was amplified by RT-PCR. Confirmed by auto-sequencing, the target gene fragment was subcloned into expression vector pET28a and transformed into E. coli BL21(DE3). With the induction of IPTG, a new recombinant protein with a relative molecular mass of 70kD appeared as the expected size and mainly existed in the soluble form. A 45kD portion of truncated Nurr1 protein was also expressed unexpectedly, and was located in inclusion bodies. Meanwhile, a 35 S-labeled recombinant Nurr1 protein was also produced using the coupled in vitro transcription and translation system in rabbit reticulocyte lysates with pET28-Nurr1 as template. These expressed 6×His-Nurr1 fusion proteins were analyzed by immunoblot with anti-Nurr1 polyclonal antibody or autoradiography, and purified by Ni-NTA affinity chromatography. In addition, one variant cDNA clone of Nurr1 was obtained from RT-PCR products, which had a deletion of 350bp within the corresponding area of exon3 and resulted in a frame shift generating a stop codon just after the deletion. Thus this variant was expected to encode a 61-amino acid residues only with a truncated N-terminal region. Whether it was produced from alternative splicing or PCR amplification remains to be determined.