基因治疗帕金森病的分子生物学研究:神经营养因子Neurturin基因克隆及其真核表达  

Study of molecular biology on gene treatment for Parkinson disease:clone of human neurturin gene in neurotrophic factor and expression of 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,北京市100054

出　　处：《中国临床康复》2005年第5期64-66,共3页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金(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：目的:克隆中国人Neurturin基因,重组携带Neurturin基因的真核表达载体,为研究Neurturin基因治疗帕金森病提供分子生物学基础。方法:实验于2003-03/2004-05在北京市神经再生修复研究重点实验室完成。采用RT-PCR方法从人胎儿脑中获取NeurturincDNA,将其克隆至pGEM-Easy-T载体中。测序鉴定后,重组携带Neurturin基因的反转录病毒载体pLPCX-Neurturin;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:RT-PCR扩增到人NeurturincDNA片段,序列与Genebank登录的序列一致;将Neurturin基因亚克隆至反转录病毒载体得到插入方向正确的重组载体pLPCX-Neurturin,病毒上清滴度为5×104CFU/mL。结论:获得人Neurturin基因cDNA序列,检测到Neurturin基因在真核细胞中表达,并得到较高滴度的病毒上清。AIM:To clone the Chinese human gene of neurturin,in and reorganize expression vector of eukaryotic cells in order to provide molecular biology base for studying the neurturin gene in treating Parkinson disease(PD). METHODS:The experiment was finished in Beijing Key Laboratory of Neural Regeneration and Repairing between March 2003 and May 2004.Total cellular neurturin cDNA was isolated from human fetal brain and cloned into pGEM Easy T with RT PCR.The retroviral vector pLPCX Neurturin with neurturin gene was reorganized after sequencing.The recombinant pLPCX Neurturin were transferred into PT67 cell lines by liposomes,and the viral tite of supernatant was determined by transfected NIH 3T3 cell lines with serial dilutions of viral supernatant. RESULTS:The RT PCR amplified human neurturin cDNA fragment was matched with the expected sequence in Genebank.The recombinant pLPCX Neurturin with right direction was obtained by subcloning Neurturin gene into retroviral vector and the viral tite was 5×104 CFU/mL. CONCLUSION:The results show that the full length cDNA of human neurturin gene is cloned and the expression in eukaryotic cells can be estimated. Also, higher tite of viral supernatant is obtained.

关 键 词：帕金森病 克隆 分子 基因表达 

分 类 号：R742.5[医药卫生—神经病学与精神病学]