人BDNF的克隆、表达及其活性检测  

GENE CLONING, EXPRESSION AND BIOLOGICAL ACTIVITY ANALYSIS OF HUMAN BRAIN DERIVED NEUROTROPHIC FACTOR

作　　者：左 皓[1] 谢佐平[1] 孙朝晖[1] 李 鹏[1] 赖燕来[1] 

机构地区：[1]清华大学 生物科学与技术系 神经生物学实验室,北京100084

出　　处：《神经解剖学杂志》2004年第6期543-547,共5页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(No.30070245)资助项目

摘　　要：为了研究脑源性神经营养因子在大肠杆菌中的表达及其在治疗Parkinson病中的作用,采用RT-PCR技术,从人胚脑组织扩增及克隆了BDNF全长及其成熟蛋白编码基因,经测序证实后,将其分别克隆至表达载体pcDNA3.1与pGEX6p-1,构建了重组真核表达载体pcDNA3.1-BDNF和重组原核表达载体pGEX6p-BDNFs。pcDNA3.1-BDNF经脂质体介导转染至体外培养的E16胚鼠中脑星形胶质细胞,以G418筛选出抗性阳性克隆,该克隆株与E12-E14胚鼠中脑神经干细胞体外共培养,酪氨酸羟化酶免疫细胞化学反应显示,转染细胞株所表达的BDNF蛋白具有生物学活性,可延长前体细胞的存活并促进其向多巴肢神经元分化。将pGEX6p-BDNFs转化大肠杆菌,以IPTG诱导,获得了相对分子质量约为40 kD的GST-BDNF融合蛋白,其表达量约占菌体总蛋白的10％,并经免疫印迹证实具有特异的免疫反应性。In order to study the expression of BDNF in E. Colt and further investigate its therapeutical effects on Parkinson's disease, we amplified the full-length and mature cDNA fragments of the human BDNF coding region from human fetal brain tissue by RT-PCR method. Confirmed by sequencing, these target gene fragments were subcloned respectively into expression vector pcDNA3. 1 and pGEX6p-l. The recombinant eukaryotic expression vector pcDNA3. 1-BDNF was transfected into E16 rat mesencephalic astroglia with lipofectamine. The human rBDNF secreted by transfected astroglia can promote the development and survival of co-cultured mesencephalic progenitors from E12-14 rat embryos. The recombinant prokaryotic expression vector pGEX6p-BDNFs was transformed into E. colt DH5a. With induction of IPTG, a new protein with relative molecular mass of 40000 was expressed expectedly and mainly located in inclusion bodies, representing 10% of total bacterial protein in E. colt. These expressed GST-BDNF fusion proteins were identified by immunoblot with anti-BDNF antibody.

关 键 词：脑源性神经营养因子 原核表达 星形胶质细胞 多巴胺神经元 克隆 人 

分 类 号：R346[医药卫生—基础医学]