人β神经生长因子基因真核表达载体的构建与表达

目的 构建人NGF-β基因真核表达载体并观察其在子鼠脊髓神经干细胞内的表达.方法 应用逆转录-聚合酶链反应(RT-PCR)从人脑肿瘤旁组织总RNA中扩增出750 bp片段,将其克隆至真核表达载体pcDNA3中经酶切鉴定产生750 bp和5.2 kd)的片段,完成序列分析.分离培养E14子鼠脊髓神经干细胞,以非脂质体转染试剂FuGENE HD介导质粒peDNA3-hNGFb转染培养第3代的细胞,应用免疫细胞化学和Western blot鉴定NGF-β在细胞内的表达.结果 RT-PCR产物为750 bp的片段,重组质粒peDNA3-hNGFb经双酶切产生750 bp和5.2kd)的片段,测序结果与文献报道结果完全一致.免疫细胞化学、Western blot结果表明NGF-β能在细胞中正确表达.结论 成功构建了peDNA3-hNGFb真核表达载体,其转染的子鼠脊髓神经干细胞能正确表达NGF-β.

Construction and expression of recombinant eukaryotie expression vector human NGF-β gene in spinal cord-derived neural stem cells

Objective To construct the eukaryotic expression vector pcDNA3-hNGFb and investigate its expression in spinal cord-derived neural stem cells. Methods The 750 bp fragment was amplified by RT-PCR from human brain tissue. By gene recombination technique, the fragments were inserted into eukaryotie expression vector pcDNA3. The recombinant plasmid was identified with restriction enzyme to digest into 750 bp and 5.2 kb fragments and DNA sequencing. The El4 rat embryonic spinal cord-derived neural stem cells were isolated and cultured. The cells of the third passage were transfected with plasmid PeDNA3-hNGFb using FuGENE HD transfection reagent. The expression of NGF-β was analyzed by immunocytochemistery as well as Western blot. Results The RT-PCR product of NGF-β coding sequence was 750 bp specific fragment. By restriction enzyme digestion, the recombind plasmid vector was digested into 750 bp and 5.2 kb fragments. The immunocytochemistery and Western blot showed the NGF-β was expressed successfully in spinal cord-derived neural stem ceils. Conclusion The eukaryotic expression vector pcDNA3-hNGFb was constructed successfully and the NGF-β gene can be expressed successfully in spinal cord-derived neural stem cells.