人脑源性神经营养因子及神经营养素3重组腺病毒的构建及表达  

Construction and Characterization of Recombinant Adenoviruses Expressing Biologically Active Human Brain derived Neurotrophic Factor and Neurotrophin-3

作　　者：陈谦[1] 汪家政[1] 刘红[1] 吴燕[1] 范明[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850

出　　处：《生物化学与生物物理学报》2000年第2期121-125,共5页

基　　金：国家自然科学基金!No .3 9770 2 5 7&&

摘　　要：为了有效的将脑源性神经营养因子 (BDNF)和神经营养素 3 (NT3 )送入神经系统 ,以达到对神经系统退行性疾病和神经损伤的治疗目的 ,构建了人BDNF和NT3重组腺病毒。首先构建了带有CMV BDNF BGHpA和CMV NT3 BGHpA表达盒的腺病毒穿梭表达载体 ,与pJM17共转染 2 93细胞后经同源重组获得了人BDNF和NT3重组腺病毒。经PCR鉴定后 ,CsCl超离心纯化获得了滴度分别为 1× 10 12 和 8× 10 11的BDNF和NT3重组腺病毒。为了检测BDNF和NT3基因的表达 ,HeLa细胞在感染后 3d ,提取细胞总RNA进行RT PCR反应 ,Wes tern印迹和ELISA分析检测外源基因的表达产物 ,结果显示BDNF和NT3基因在重组腺病毒中可有效的转录和翻译。同时感染后的HeLa细胞上清可有效的促进八日龄鸡胚背根神经节组织块突起的生长 ,这说明表达的蛋白质具有良好的生物学活性。In order to deliver brain derived neurotrophic factor(BDNF) and neurotrophin 3(NT3) into CNS to prevent or reduce degeneration of CNS neurons in human neurodegenerative diseases and neuron injuries, recombinant adenoviruses Ad BDNF and Ad NT3 were constructed. BDNF and NT 3 genes were inserted into an E1 substituted adenovirus shuttle plasmid, respectively, and were driven by the human cytomegalovirus immediate early gene promoter/enhancer. After cotransfection into 293 cells with the adenovirus plasmid pJM17, the recombinant adenovirus Ad BDNF and Ad NT3 were propagated in 293 cells via homologous recombination. After two rounds of CsCl centrifugation, the human recombinant adenovirus Ad BDNF and Ad NT3 were obtained with titer of 1×10 12 and 8×10 11 pfu/ml, respectively. To examine the expression of BDNF and NT3, HeLa cells were infected with Ad BDNF and Ad NT3, respectively. RT PCR, Western blot and ELISA analysis results showed that BDNF and NT 3 genes could be transcribed and translated in Ad BDNF and Ad NT3 infected HeLa cells. And the culture media containing 10% conditioned medium of Ad BDNF and Ad NT3 infected Hela cells could induce the neurite outgrowth from E8 dorsal root ganglion neurons.

关 键 词：BDNF NT3 重组腺病毒 基因表达 生物学活性 

分 类 号：Q786[生物学—分子生物学]