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利用体内同源重组原理,构建了能介导GDNF基因转移和表达的复制缺陷型重组腺病毒AdCMVgdnf,其中GDNFcDNA插入腺病毒基因组的E1区并由CMV启动子控制在人293细胞内通过同源重组包装生成重组腺病毒后,用形态学方法、病毒DNA酶切分析、PCR和RT-PCR等方法进行鉴定正确.经测定病毒滴度达到1010pfu/ml.用免疫沉淀方法从重组腺病毒感染的293细胞及其培养基上清中均检测到大量GDNF蛋白.用重组腺病毒直接感染或者用其条件培养基处理,分别使胚胎大鼠中脑原代多巴胺能神经元的数目增加88.2%和96.4%,明显增加多巴胺能神经元存活,对帕金森氏病基因治疗具有重要意义
Using homologous recombination in vivo, a replication-defective recombinant adenovirus AdCMVgdnf that can mediate GDNF gene transfer and expression was constructed, where GDNF cDNA was inserted into the E1 region of the adenovirus genome and controlled by the CMV promoter in human 293 cells by homologous recombination After the recombinant adenoviruses were packaged, the methods of morphology, viral DNA digestion, PCR and RT-PCR were used to identify the recombinant adenovirus. The measured virus titer reached 1010pfu / ml. A large number of GDNF proteins were detected by immunoprecipitation from recombinant adenovirus-infected 293 cells and their culture supernatants. Direct infection with recombinant adenovirus or its conditioned medium, respectively, increased the number of primary mesencephalic dopaminergic neurons in embryonic rats by 88.2% and 96.4%, significantly increasing the survival of dopaminergic neurons, Gene therapy of Parkinson’s disease is of great importance