【摘要】 目的构建大鼠神经营养因子3(NT-3)基因真核表达载体并观察其在真核细胞内的表达情况。方法采用逆转录聚合酶链式反应(RT-PCR)技术从大鼠脑组织总RNA中扩增NT-3基因cDNA序列,将其克隆到真核表达载体pcDNA3中,经酶切鉴定和序列分析后,以阳离子脂质体Li-pofectamine2000介导转染L929细胞,应用免疫细胞化学和western blot鉴定NT-3在细胞内的表达。结果RT-PCR产物为822bp的特异片段,重组质粒pcDNA3/NT-3酶切后产生822bp和5.2kb的片段,DNA测序证实822bp片段的碱基序列与大鼠NT-3基因序列完全一致,成功构建了pcDNA3/NT-3重组质粒。将其转染真核细胞后,免疫细胞化学、western blot结果表明NT-3能在真核细胞中正确表达。结论成功构建了重组真核表达质粒载体pcDNA3/NT-3,为后续的研究奠定基础。更多还原

【Abstract】 Objective To construct the eukaryotic expression recombinant plasmid pcDNA3/NT-3 and investigate its expression in L929 cells.Methods The gene of rat neurotrophin 3(NT-3)was amplified by RT-PCR from rat brain tissue.By gene recombination technique,rat NT-3 coding sequence was inserted into eukaryotic expression vector pcDNA3.The recombinant plasmid was verified with restriction enzyme digestion and DNA sequencing.L929 cells were transfected with the recombinant vector using Lipofectamine 2000 transfection reagent.The expression of NT-3 was analyzed by immunocytochemistery as well as western blot.Results The RT-PCR product is 822bp specific segment.By restriction enzyme digestion,the recombinant plasmid was digested into 822bp and 5.2kb fragments.The DNA sequence of the 822bp fragment was identical with rat NT-3 cDNA in GeneBank.The immunocytochemistery and western blot showed the NT-3 was expressed successfully in L929 cells.Conclusions The recombinant plasmid pcDNA3/NT-3 was constructed successfully,which will provide the foundation for the further research.更多还原