大鼠脑源性神经营养因子基因重组真核表达载体的构建及其表达  

Construction and expression of gene recombinant eukaryotic expression vector carrying rat brain-derived neurotrophic factor

作　　者：邓兴力[1] 杨智勇[1] 董坚[2] 王廷华[3] 徐丹[3] 冯忠堂[3] 

机构地区：[1]昆明医学院第一附属医院神经外科,云南省昆明市650032 [2]昆明医学院第一附属医院生物治疗中心,云南省昆明市650032 [3]昆明医学院神经科学研究所,云南省昆明市650031

出　　处：《中国组织工程研究与临床康复》2008年第28期5473-5476,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:脑源性神经营养因子对多种神经元的发育、存活及轴突再生中起着重要作用,但由于血脑屏障的存在,限制了其应用。目的:构建大鼠脑源性神经营养因子基因重组真核表达载体,观察其在真核细胞内的表达。设计、时间及地点:单一样本实验,于2005-09/2006-09在昆明医学院神经科学研究所完成。材料:清洁级健康雄性8周龄SD大鼠,体质量250g。方法:①以反转录-聚合酶链反应从大鼠脑组织总RNA扩增脑源性神经营养因子基因编码序列,将其克隆到真核表达载体pcDNA3中,构建重组表达载体pcDNA3/BDNF。②将L939细胞分为pcDNA3/BDNF转染组、pcDNA3转染组和未转染组,以FuGene HD转染试剂介导相应质粒转染。主要观察指标:①重组质粒pcDNA3/BDNF的酶切鉴定与序列分析。②免疫细胞化学和western blot鉴定脑源性神经营养因子在L929细胞中的表达。结果:①重组质粒pcDNA3/BDNF经酶切产生783bp和5.2kb的片段,DNA测序证实783bp片段的碱基序列与大鼠脑源性神经营养因子基因编码序列完全一致。②基因转染后,免疫细胞化学、western blot结果表明脑源性神经营养因子能在真核细胞中正确表达。结论:成功构建了重组真核表达质粒载体pcDNA3/BDNF,为后续研究奠定了基础。BACKGROUND： Brain-derived neurotrophic factor （BDNF） plays an important role in the development and survival of varied neurons and axonal regeneration. However, blood-brain barrier limits its application. OBJECTIVE： To construct the eukaryotic expression recombinant plasmid of rat brain-derived neurotrophic factor, and to investigate its expression in eukaryocytes. DESIGN, TIME AND SETTING： The single sample experiment was performed at the Institute of Neuroscience, Kunming Medical College from September 2005 to September 2006. MATERIALS： Healthy 8-week male Sprague Dawley （SD） rats of clean grade and weighing 250 g were used in this study. METHODS： The coding sequence of rat brain derived neurotrophic factor was amplified by reverse transcription-polymerase chain reaction （RT-PCR） from total RNA of rat brain tissues. Brain-derived neurotrophic factor coding sequence was inserted into eukaryotic expression vector pcDNA3. The recombinant plasmid pcDNA3/BDNF was verified. L929 cells were assigned into pcDNA3/BDNF-transfection group, pcDNA3-transfection group and non-transfection group. Corresponding plasmid was transfected with FuGene HD transfection reagent. MAIN OUTCOME MEASURES： Enzyme digestion and sequencing of recombinant plasmid; The expression of brain-derived neurotrophic factor was analyzed by immunocytochemistery and western blot. RESULTS： By restriction enzyme digestion, the recombinant plasmid pcDNA3/BDNF was digested into 783 bp and 5.2 kb fragments. The DNA sequence of the 783 bp fragments was identical with rat brain-derived neurotrophic factor cDNA in GeneBank. The immunocytochemistery and western blot showed brain-derived neurotrophic factor was expressed successfully in L929 cells. CONCLUSION： The recombinant plasmid pcDNA3/BDNF is constructed successfully, which will provide the foundation for the further research.

关 键 词：脑源性神经营养因子 基因表达 质粒 转染 组织构建 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]