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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
OBJECTIVE: To construct the eukaryotic expression vector of mouse β NGF gene and observe the expression of NGF in NIH 3T3 cell line stably transfected with it. Methods: The mature cDNA sequence of mouse β NGF and its leader sequence were cloned into the eukaryotic expression vector pcDNA3.1 + by gene recombination technique, and the recombinant was identified by restriction enzyme digestion. NIH 3T3 cells were transfected with the lipofectamine FuGENETM6 method. At 72h after transfection, positive clones were screened with G418 and grown until 20 days. The expression of NGF in the positive clone culture supernatant was analyzed by Western blot and the effect of NGF in the supernatant on the prominence of PC12 cells was observed. Results: The β NGF gene was expressed in NIH 3T3 cells. Positive clonal culture supernatant can promote PC12 cell protrusion growth. CONCLUSION: The recombinant eukaryotic expression vector pcDNA3.1 + / NGF has been successfully constructed. NGF protein is expressed in NIH 3T3 cells and has good biological activity, which lays the foundation for the further development of NGF gene therapy for nervous system diseases