【摘要】 目的构建含有大鼠脑源性神经营养因子(BDNF)基因的重组腺病毒(AD)载体,并分析其体外表达情况。方法将采用RT-PCR技术获取的大鼠BDNF的cDNA基因定向克隆入穿梭质粒pAdTrack-CMV中。通过与腺病毒骨架载体pAdeasy-1在细菌内同源重组形成重组腺病毒质粒pAd-BDNF,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(Ad-BDNF)。重组病毒感染体外培养的Hela细胞后,用RT-PCR、Western blot及免疫细胞化学检测细胞BDNF基因及蛋白表达情况。结果 pAd-BDNF测序结果和预期一致,同源重组并经293细胞包装后形成重组腺病毒Ad-BDNF,重组病毒感染Hela细胞后能高水平表达BDNF。结论成功制备了重组腺病毒Ad-BDNF,感染细胞证实其呈分泌表达,为将其进一步应用于神经系统损伤性疾病治疗的研究打下了基础。更多还原

【Abstract】 Objective To construct adenovirus vector encoding rat brain derived neurotrophic factor(BDNF) gene and identify the expression in vitro.Methods The specific BDNF sequence was cloned into the plasmid of pAdTrack-CMV to construct the BDNF expression plasmid pAd-BDNF.The recombinant plasmids were identified by DNA sequencing and restriction digestion.The homologous recombination between the recombinant vector and the adenovirus bone vector pAdeasy-1 in the Ecoli BJ5183 resulted to the formation of recombinant adenovirus vector Ad-BDNF.The infecting recombinant virus particles Ad-BDNF was produced after the recombinant adenovirus vector had been transfected to the human embry kidney 293 cells.The recombinant adenovirus vector infected the hela cell,the expression of BDNF was detected by RT-PCR,Western blot and immunocytochemistry.Results The sequence results of pAd-BDNF were consistent with expectancy.After homologous recombination and packaging with 293 cells,the recombinant Ad-BDNF adenovirus was obtained.RT-PCR,Western blot and immunocytochemistry suggested that the BDNF was expressed.Conclusions The Ad-BDNF was constructed successfully.It is confirmed that the interest proteins were expressed in the infected cells,which lays the basis for its application in the treatment of the neurodamaged diseases.更多还原