人Nurr1基因克隆及其在真核细胞中的表达  

CLONING AND EXPRESSION OF HUMAN NUCLEAR RELATED RECEPTOR 1(Nurr1) GENE IN EUKARYOTIC CELLS

作　　者：张京钟[1] 余爽[1] 段德义[1] 赵春礼[1] 徐群渊[1] 

机构地区：[1]首都医科大学北京神经科学研究所,北京市神经再生修复研究重点实验室,北京100054

出　　处：《神经解剖学杂志》2005年第3期247-251,共5页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(No.30270433);北京市教育委员会科技发展计划(KM200310025092);北京市科技新星计划(020820650120);北京市优秀人才专项经费资助项目。

摘　　要：为探讨Nurr1基因治疗Parkinson病的可行性,本研究克隆了中国人核相关受体1(nuclearrelatedreceptor1,Nurr1)基因,并重组携带Nurr1基因的真核表达载体,为该研究提供分子生物学基础。实验采用反转录聚合酶链式反应(RTPCR)方法从人胎儿脑中获取Nurr1cDNA,将其克隆至pGEMTEasy载体中;测序鉴定后,重组携带Nurr1基因的逆转录病毒载体pLNCX2Nurr1;通过脂质体将重组载体转染PT67包装细胞系,用病毒上清感染NIH3T3细胞系以检测病毒滴度。结果发现,RTPCR扩增得到人Nurr1cDNA片段,序列与GenBank登录的序列一致;将Nurr1基因亚克隆至逆转录病毒载体得到插入方向正确的重组载体pLNCX2Nurr1,病毒上清滴度为2×105CFU/ml。结果表明本实验获得人Nurr1基因cDNA序列,检测到Nurr1基因在真核细胞中表达,并得到较高滴度的病毒上清。To obtain the Chinese human gene of Nurr1 for the treatment of Parkinson's disease, total cellular RNA was isolated from human fetal brain and reversely transcribed into cDNA by reverse transcription-polymerase chain reaction (RT-PCR) . The correct Nurr1 cDNA was subcloned into retrovirus vector pLNCX2. The recombinant pLNCX2-Nurr1 was transferred into PT67 package cell lines by Lipofectamine^TM 2000, and the viral titer of supernatant was determined by transfecting NIH 3T3 with serial dilution method. The results showed that the target human Nurr1 cDNA fragment matched the expected sequence in GenBank. The recombinant pLNCX2-Nurr1 could express Nurr1 gene in PT67 cell lines and the viral titer was 2×105 CFU/ml. The results suggested that the full-length cDNA of Chinese Nurr1 was cloned and could be expressed in eukaryotic cells.

关 键 词：人Nurr1基因 基因克隆 基因表达 帕金森病 基因治疗 

分 类 号：R742.5[医药卫生—神经病学与精神病学] R394.8[医药卫生—临床医学]