Objective: A recombinant replication deficient adenvirus vector which codes for wild-type mouse npcl gene was constructed for the therapeutic strategy of Niemann-Pick disease type C. Methods: The wild-type npcl was extracted from prp-pro-mnpcl with HindⅢ and kpn I, and then inserted to the E1 deleted expression plasmid padtrack-cmv shuttle vector, named PAd-track-cmv. The construct was cotransfected with the 293 cells by liposome-modiated method. The recombinant adenovirus was detected by examining the expression of a green fluorescence protein tag in the 293 cells. Results: By sequencing, it was confirmed that the product was the favorite gene of npcl. Restriction endonuclease analysis confirmed the successful cloning of the fusion gene into to pAdTrackCMV. The recombinants (pAd-npcl) were selected for kanamycin resistance, and recombination was confirmed by restriction endounuclease analysis. Presence of the recombinant adenoviruses was confirmed by GFP expression.Conclusions: The recombinant adenovirus vector of the npcl was constructed successfully, which is helpful to the further investigation of its potentiality in the study of Niemann Pick's disease type c.%目的以复制缺陷的腺病毒为载体,细菌内同源重组法构建有npcl基因的腺病毒载体.方法利用限制性内切酶kpnl HindⅢ从带有朊病毒启动子的质粒prp-pro-mlpcl中切下目的基因npcl-1,克隆至穿梭质粒PAdtrack-CMV,再与PAdeasy-1在大肠杆菌BJ5183中进行同源重组,经抗性筛选和酶切鉴定的质粒再利用脂质体转染人293例细胞中扩增,利用Adeasy-1系统上的绿色荧光蛋白鉴定病毒,RT-PCR鉴定目的基因的表达.结果切酶切鉴定,正确的目的基因片断已克隆至穿梭质粒PAdtrack-CMV;卡那霉素进行抗性筛选及Pac Ⅰ酶切鉴定证实腺病毒重组质粒构建成功.Pac Ⅰ酶切线性化的重组质粒导入293细胞3d后见明显的绿色荧光蛋白表达.回收病毒,可重复感染293细胞,证实有感染能力的病毒颗粒包装成功.结论利用腺病毒载体系统Adeasy-1可构建能成功表达Pac Ⅰ基因的腺病毒载体,为对Niemann pick' s病C型的进一步研究提供了条件.
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