人神经营养素-3基因的克隆、表达及其活性检测

为了获取神经营养素 -3 (NT-3 )将之用于神经系统疾病的治疗 ,采用 RT-PCR技术 ,从人胎脑组织特异性扩增并克隆了人 NT-3全长以及成熟蛋白编码基因 ,将其分别克隆至表达载体 pc DNA3 .1与 p ET2 8a,构建了重组真核表达载体 pc DNA3 .1-NT3和重组原核表达载体 p ET2 8-NT3 s。 pc DNA3 .1-NT3经脂质体介导转染至大鼠胚胎脑纹状体神经胶质细胞 ,以 G418筛选出抗性阳性克隆 ,该克隆株与大鼠胚脑纹状体神经干细胞体外共培养 5 d,MAP-2 (微管相关蛋白 2 )免疫细胞化学反应结果显示 ,转染细胞株所表达的 NT-3蛋白具有生物学活性 ,可促进共培养的纹状体神经干细胞的突起生长。将 p ET2 8-NT3 s转化大肠杆菌 ,以 IPTG诱导 ,获得了相对分子质量约 18k D的 6XHis-NT3融合蛋白 ,其表达量占菌体总蛋白的 2 0 %左右。经镍柱亲和纯化 ,得到纯度达 95 %的 rh NT-3蛋白 ,免疫印迹证实所获蛋白产品具有特异的免疫反应性

GENE CLONING,EXPRESSION AND BIOLOGICAL ACTIVITY ANALYSIS OF HUMAN NEUROTROPHIN-3

To obtain Neurotrophin 3 and further investigate its therapeutical effects on neurodegenerative disorders, we amplified the full length and functional cDNA fragments of the human NT 3 coding region from human fetal hippocampal tissue by RT PCR method. Confirmed by sequencing, these target gene fragments were subcloned respectively into expression vector pcDNA3.1 and pET28a. The recombinant eukaryotic expression vector pcDNA3.1 NT3 was transfected into E16 rat striatal neuroglial cells with lipofectamine, and the expression product of G418 resistant cell clones analyzed by RT PCR could promote neurite outgrowth of E13 rat striatal neural stem cells co cultured with it. The recombinant prokaryotic expression vector pET28 NT3s was transformed into E. coli BL21(DE3). With induction of IPTG, a new protein with relative molecular mass of 18 000 was expressed expectedly and mainly located in inclusion bodies, representing 20% of total bacterial protein in E.coli. These expressed 6XHis NT3 fusion proteins were identified by immunoblot with anti NT3 monoclonal antibody, and purified by Ni NTA affinity chromatography column.