CAJ | 학술논문

核受体相关因子1(nuclear receptor-related factor 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Nurr1 Cdna,并获得一个在氨基端缺失350 bp碱基的Nurr1突变体.将正常的Nurr1基因片段亚克隆至表达载体pET28a,分别在TNTRT7偶联网织红细胞溶胞系统和大肠杆菌BL21(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35S.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.
핵수체상관인자1(nuclear receptor-related factor 1,Nurr1)시주요표체우중뇌흑질급복측피개구다파알능신경원적일충전록인자,속우핵수체초가족성원,기공능성배체상미피학인.연구표명,Nurr1대중뇌다파알신경원적발육、존활이급성숙후공능적유지구유특수중요의의.여능조도타적특이성배체,장위최종사선출치료파금삼병등중추다파알실조성질병적약물혹화학합성선도물타하기출.위료획취Nurr1단백이표정기배체이급연구단백질간적상호작용,채용RT-PCR기술,종인배중뇌조직특이성확증급극륭료인Nurr1 Cdna,병획득일개재안기단결실350 bp감기적Nurr1돌변체.장정상적Nurr1기인편단아극륭지표체재체pET28a,분별재TNTRT7우련망직홍세포용포계통화대장간균BL21(DE3)중획득표체,균이가용성형식존재,차산자우체외전록/번역계통적진핵표체Nurr1단백이표기상동위소35S.Western인적분석표명，소표체적중조목적단백구유특이적면역반응성.경Ni-NTA친화층석，득도료초보순화적rhNurr1단백.
In order to obtain Nurrl protein for further ligand screening, protein-protein interaction studying and protein structure analyzing, the full-length fragment of the human Nurrl 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 70 kD appeared as the expected size and mainly existed in the soluble form. A 45 kD portion of truncated Nurrl protein was also expressed unexpectedly, and was located in inclusion bodies. Meanwhile, a 35 S-labeled recombinant Nurrl 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 350 bp 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.