军事医学2017，Vol.41Issue(12)：962-967,6.DOI:10.7644/j.issn.1674-9960.2017.12.004
利用Red重组系统在腺病毒大质粒上快速实现单碱基点突变
A highly efficient approach to introduction of the single-base mutation in a plasmid containing adenovirus genome through Red recombination
摘要
Abstract
Objective To construct a highly efficient approach to the introduction of the single-base mutation in a plasmid containing the adenovirus whole genome larger than 40 kb.Methods The target DNA with a mutation site was achieved by over-lapping PCR.The large plasmid with adenovirus genome and target DNA were co-transformed into Escherichia coli strain DY330 carrying a high rate Red recombination system.The positive clone was selected via colony PCR in combination with enzyme identification.The site-mutation large plasmid was transformed into E.coli strain DH10B in which the backbone of the large plasmid remained was stable.Results Two mutations were continuously introduced into the adenovirus genome,the location of which was pos.9171 and pos.24410 respectively.The integrality and stability of the plasmid backbone were verified by enzyme cutting identification.The two mutations on the plasmid were verified by DNA sequencing.Conclusion An efficient approach to the introduction of the single-base mutation in positions 9171 and 24410 from the adenovirus genome which was integrated into a plasmid is successfully established.The positive selection efficiency ranges from 5%to 15%.The construction of the approach will facilitate the study of adenovirus infection mechanism.关键词
Red重组/腺病毒科/质粒/定点突变Key words
Red recombination/adenovidae/plasmid/site-directed mutation分类
生物科学引用本文复制引用
羊嬉春,王芃,杨巧玲,周建光,王健,黄芳,李山虎,郑继平..利用Red重组系统在腺病毒大质粒上快速实现单碱基点突变[J].军事医学,2017,41(12):962-967,6.基金项目
国家科技重大专项课题资助项目(2014ZX09J14105-070) （2014ZX09J14105-070）
国家自然科学基金资助项目(31460699) （31460699）
海南省自然科学基金创新研究团队资助项目(2017CXTD005) （2017CXTD005）
海南省自然科学基金面上资助项目(317071) （317071）
