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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PacI酶切鉴定证实腺病毒重组质粒构建成功。PacI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PacI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。
Objective To construct a recombinant adenovirus vector containing npcl gene by homologous recombination in bacteria. Methods The target gene npcl-1 was excised from plasmid prp-pro-mlpcl with prion promoter by restriction endonuclease kpnl HindⅢ, cloned into the shuttle plasmid PAdtrack-CMV, and then ligated with PAdeasy-1 in E. coli BJ5183 After homologous recombination, plasmids identified by resistance screening and restriction enzyme digestion were amplified by lipofectin in 293 cells, identified by green fluorescent protein on Adeasy-1 system, and identified by RT-PCR expression. Results The recombinant plasmid was identified by restriction enzyme digestion. The correct target gene fragment was cloned into the shuttle plasmid PAdtrack-CMV. The kanamycin resistance screening and PacI restriction enzyme digestion confirmed the successful construction of the adenovirus recombinant plasmid. PacI restriction enzyme digestion linearized recombinant plasmid into 293 cells after 3 days to see the obvious green fluorescent protein expression. Recover the virus, 293 cells can be repeatedly infected, confirmed the ability to infecting the virus particles packed successfully. Conclusion The recombinant adenovirus vector expressing PacI gene can be constructed by adenovirus vector system Adeasy-1, which can be used for further research on C-type of Niemann pick’s disease.