pcDNA3-hNGFb真核表达载体的构建及其表达  

Construction of recombinant plasmid pcDNA3-hNGFb and its expression

作　　者：邓兴力[1] 杨智勇[1] 董坚[2] 王廷华[3] 徐丹[3] 冯忠堂[3] 

机构地区：[1]昆明医学院第一附属医院神经外科,650032 [2]昆明医学院第一附属医院生物治疗中心,650032 [3]昆明医学院神经科学研究所

出　　处：《神经疾病与精神卫生》2008年第2期114-117,共4页Journal of Neuroscience and Mental Health

摘　　要：目的构建人神经生长因子β(NGF-β)基因真核表达载体并观察其在L929细胞内的表达。方法以RT-PCR从人脑组织总RNA中扩增NGF-βcDNA,将其克隆到真核表达载体pcD-NA3中,经酶切鉴定和序列分析后,以Lipofectamine2000介导转染L929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750bp的特异片段,重组质粒pcDNA3-hNGFb酶切后产生750bp和5.2kb的片段,DNA测序证实750bp片段的碱基序列与人NGF-βcDNA完全一致。将其转染L929细胞后,免疫细胞化学、western blot结果表明NGF-β及其前体proNGF-β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pcDNA3-hNGFb,为后续的研究奠定基础。Objective To construct the eukaryotic expression recombinant plasmid pcDNA3- hNGFb and investigate its expression in L929 cells. Methods The cDNA of human nerve growth factor beta subunit （NGF-β） was amplified by RT-PCR from human brain tissue. By gene recombination technique, human NGF-β cDNA was inserted into eukaryotic expression vector pcDNA3. The recombinant plasmid was verified with restriction enzyme digestion and DNA sequencing. Transfected the recombinant vector into L929 cells with Lipofectamine 2000 transfection reagent. The expression of NGF-β was analyzed by immunocytochemistery as well as western blot. Results The RT- PCR product is 750bp specific segment. By restriction enzyme digestion, the recombinant plasmid was digested into 750bp and 5.2kb fragments. DNA sequence result showed the 750bp fragment was identical with human NGF-β cDNA in GenBank. Immunocytochemistery and western blot showed the NGF-β was expressed successfully in L929 cells. Conclusions The recombinant plasmid pcDNA3 - hNGFb was constructed successfully, which will provide the foundation for further research.

关 键 词：神经生长因子 真核表达 重组质粒 转染 

分 类 号：R346[医药卫生—基础医学]