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目的构建具有生物活性的人神经营养素_3(NT_3)基因重组腺病毒表达载体。方法从人脑组织mRNA中扩增NT_3基因全长cDNA,定向克隆于穿梭质粒pShuttle中,获得一个带有CMV启动子的质粒。再与腺病毒骨架DNA(Adeno-X viral DNA)体外连接,形成重组腺病毒质粒(pAd_NT_3)。用pAd_NT_3转染人胚肾293细胞后包装成有感染能力的重组腺病毒颗粒(Adeno_NT_3)。结果NT_3基因RT-PCR扩增产物约801 bp。Adeno_NT_3 PCR鉴定为正确重组子。RT_PCR、免疫细胞化学、ELISA及Western blot检测显示,Adeno_NT_3能在其转染的293细胞表达和分泌NT_3。这种NT_3能使体外培养的神经干细胞更多地分化为神经元样细胞。结论应用体外连接法成功构建了人NT_3基因重组腺病毒表达载体,其表达产物具有促进神经干细胞分化为神经元样细胞的活性作用。
Objective To construct a biologically active human neurotrophic factor-3 (NT_3) gene recombinant adenovirus expression vector. Methods The cDNA of NT_3 gene was amplified from human brain mRNA and cloned into shuttle plasmid pShuttle to obtain a plasmid with CMV promoter. And then ligated with adenovirus DNA (Adeno-X viral DNA) to form a recombinant adenovirus plasmid (pAd_NT_3). 293 cells were transfected with pAd_NT_3 and packaged into competent adenovirus (Adeno_NT_3). Results The RT-PCR product of NT_3 gene was about 801 bp. Adeno_NT_3 PCR was identified as the correct recombinant. RT_PCR, immunocytochemistry, ELISA and Western blot showed that Adeno_NT_3 could express and secrete NT_3 in 293 cells transfected with it. This NT_3 enables more differentiation of neural stem cells cultured in vitro into neuron-like cells. Conclusion The recombinant adenovirus vector of human NT_3 gene was successfully constructed by in vitro ligation method. The expressed product of the recombinant adenovirus was able to promote the differentiation of neural stem cells into neuron - like cells.