摘要
目的 :利用大肠杆菌BJ5 183细菌内同源重组构建重组腺病毒载体。方法 :自过氧化氢酶质粒载体pZeoSV2 -Cat中酶切出CatcDNA ,亚克隆至质粒 pBluescriptⅡsk(+)中 ,形成 pBluescriptⅡsk(+) -Cat ,自pBlue scriptⅡsk(+) -Cat中酶切出CatcDNA ,亚克隆至腺病毒穿梭质粒 pShuttle -CMV中 ,形成转移质粒 pShuttle -CMV -Cat,在感受态大肠杆菌BJ5 183内与腺病毒骨架质粒 pAdEasy - 1同源重组 ,得到重组腺病毒载体 pAdEasy- 1-Cat。对 pAdEasy - 1-Cat进行酶切鉴定和PCR鉴定。结果 :线性化的pShuttle-CMV -Cat转化含pAdEasy- 1的超感受态大肠杆菌BJ5 183,2 4h后获得了 30 %阳性重组质粒克隆 ,经酶切获得一大于 2 0kb的大片段和一4 .5kb的特征性条带 ,PCR反应扩增出 2 0 9bp片段。结论 :应用细菌内同源重组能快速构建含Cat基因的重组腺病毒载体pAdEasy - 1-Cat。
Objective To prepare Catalase-contained recombinant adenovirus vector by the homologous recombination in bacteria. Methods Catalase gene was digested from pZeoSV2-Cat, subcloned into plasmid of pBluescriptⅡsk(+) and formed plasmid of pBluescriptⅡsk(+)-Cat. Then Catalase gene was digested from plasmid of pBluescriptⅡsk(+)-Cat, subcloned into shuttle plasmid of pShuttle-CMV and formed transfer plasmid of pShuttle-CMV-Cat.Adenovirus genomic plasmid of pAdEasy-1 was transformed into BJ5183 bacteria and prepared ultracompletent BJ5183 containing pAdEasy-1. Results The linealinzed pShuttle-CMV-Cat was transformed into ultracompletent BJ5183 containing pAdEasy-1.There were 30% positive recombinant plasmid. PCR test indicated that the recombinant adenovirus plasmid pAdEasy-1-Cat contained Catalase gene. Conclusion The homologous recombination in bacteria is a convenient and efficient method to prepare recombinant adenovirus plasmid pAdEasy-1-Cat. This offers a good gene transfer vector for the gene therapy in restenosis.
出处
《郧阳医学院学报》
2003年第4期193-196,共4页
Journal of Yunyang Medical College
基金
湖北省科技厅科技攻关项目 (No .2 0 0 1AA30 7B0 7)
关键词
同源重组
腺病毒载体
基因
过氧化氢酶
基因治疗
细菌
Homologous recombination
Adenoviral vector
Gene,Catalase
Gene therapy
bacteria
