小鼠NGF基因真核表达载体的构建及其在NIH3T3细胞中的表达

目的 :构建小鼠 β NGF基因真核表达载体并观察其稳定转染的NIH 3T3细胞系中NGF的表达情况。方法 :用基因重组技术 ,将小鼠 β NGF的成熟cDNA序列及其前导序列 ,克隆到真核表达载体 pcDNA3.1+中 ,用限制性内切酶酶切鉴定重组体。利用脂质体FuGENETM6方法 ,转染NIH 3T3细胞。转染后 72h ,用G4 18筛选阳性克隆并培养至 2 0d。对阳性克隆培养上清中NGF的表达用Westernblot分析并观察该上清中NGF对PC12细胞突起生长的作用。结果 :β NGF基因在NIH 3T3细胞中获得表达。阳性克隆培养上清能促进PC12细胞突起生长。结论 :重组真核表达载体 pcDNA3.1+/NGF构建成功 ,NGF蛋白在NIH 3T3细胞中表达 ,并具有良好的生物学活性 ,为进一步开展NGF基因治疗神经系统疾病奠定了基础

Construction of the eukaryotic expression vector of mouse β-NGF and its exp ression in NIH 3T3 fibroblast cell lines

AIM: To construct the eukaryotic expression vector of mouse nerve growth factor gene and express it in NIH 3T3 fibroblast cell lines. METHODS: By gene recombination technique, mouse beta-NGF cDNA was inserted into mammlian expression vector pcDNA3.1+. The recombinant plasmid was verified with restriction enzyme digestion analysis. NIH 3T3 cell grown to log phase was transfected with this vector using FuGENE(TM) 6 transfection reagent. The transfected cells were grown in DMEM medium containing G418 at 72 hours after transfection, and the positive clones were selected in G418 medium until the 20 th day. The expression of NGF and its biological activity were analyzed by Western blot and the neurite outgrowth of PC12 cells stimulated by culture supernatant of positive clones respectively. RESULTS: The beta-NGF gene was expressed successfully in NIH 3T3 cells. The culture supernatant could stimulate the neurite outgrowth of PC12 cells. CONCLUSION: The eukaryocyte expression vector was constructed successfully. The NGF gene was expressed successfully in the transfected NIH 3T3 cells with good biological activity, which laid the foundation for gene therapy of nervous system diseases.