摘要
目的采用细菌内同源重组法高效制备含神经生长因子β基因(hNGFβ)的重组腺病毒质粒。方法hNGFβ自载体pBLAST44hNGFβv02中切出,亚克隆至质粒pBluescriptⅡsk(+)中,形成pBluescriptⅡsk(+)hNGFβ,自pBluescriptⅡsk(+)hNGFβ中酶切出hNGFβDNA,亚克隆至腺病毒穿梭质粒pShuttleCMV中,形成转移质粒pShuttleCMVhNGFβ,在感受态大肠杆菌BJ5183内与腺病毒骨架质粒pAdEasy1同源重组,得到重组腺病毒载体pAdEasy1hNGFβ。结果线性化的pShuttleCMVhNGFβ转化含pAdEasy1的高效感受态大肠杆菌BJ5183,24h后获得了30%阳性重组质粒克隆,经酶切获得大于20kb的大片段和4.5kb的特征性条带,PCR反应扩增出731bp片段。结论应用细菌内同源重组能快速构建含hNGFβ基因的重组腺病毒载体pAdEasy1hNGFβ,为hNGFβ基因的研究及应用奠定了基础。
Objective To prepare nerve growth factor(NGF)-contained recombinant adenovirus vector by the homologous recombination in bacteria.Methods NGF gene was digested from pBLAST44-hNGFβv02,subcloned into plasmid of pBluescriptⅡsk(+) and plasmid of pBluescript Ⅱsk(+)-hNGFβ formed.Then hNGFβ gene was digested from plasmid of pBluescript Ⅱsk(+)-hNGFβ,subcloned into shuttle plasmid of pShuttle-CMV and transfer plasmid of pShuttle-CMV-hNGFβ formed.Adenovirus genomic plasmid of pAdEasy-1 was transformed into BJ5183 bacteria and ultracompletent BJ5183 containing pAdEasy-1 prepared.Results The linealinzed pShuttle-CMV-hNGFβ was transformed into ultracompletent BJ5183 containing pAdEasy-1.There were 30% positive recombinant plasmids.PCR test indicated that the recombinant adenovirus plasmid pAdEasy-1-hNGFβ contained NGF gene.Conclusion The homologous reconbiation in bacteria is a convenint and efficient method to prepare recombinant adenovirus plasmid pAdEasy-1-hNGFβ.This offers a good gene transfer vector for the gene therapy in pain management.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2005年第3期287-290,共4页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
湖北省自然科学基金资助项目(No.2005ABA080)
湖北省卫生厅青年科技人才基金资助项目(No.QJX200516)
关键词
同源重组
腺病毒
人β神经生长因子
homologous recombination
adenovirus
human nerve growth factor β
