位点特异整合微环DNA的体内制备  

An in vivo Robust System for Generation of Site-specific Integration Minicircle DNA Vector

作　　者：聂永强[1] 马海燕[1,2] 马晴雯[1,2] NIE Yong-qiang MA Hal-yan MA Qing-wen(Shanghai Institute of Medical Genetics, Shanghai Children＇s Hospital, Shanghai Jiao Tong University, Shanghai 200040, China Key Laboratory of Embryo Molecular Biology, Ministry of Health ＆ Shanghai Key Laboratory)

机构地区：[1]上海交通大学附属儿童医院上海市儿童医院上海交通大学医学遗传研究所,上海200040 [2]卫生部医学胚胎分子生物学重点实验室暨上海市胚胎与生殖工程重点实验室,上海200040

出　　处：《中国生物工程杂志》2017年第7期80-87,共8页China Biotechnology

基　　金：上海市卫生局青年基金资助项目(20154Y0152)

摘　　要：目的:应用诱导表达的LR克隆酶系统,建立一种在细菌体内获得基于链霉菌噬菌体ФC31整合酶系统的位点特异整合型微环DNA的方法,为实现无细菌骨架等冗余序列的转基因奠定基础。方法:构建包含阿拉伯糖启动子的LR克隆酶系统和ФC31整合酶系统的亲本质粒,在L-阿拉伯糖的诱导下重组产生表达ФC31整合酶的微质粒和包含有目的基因和attB位点等元件的微环DNA。以限制性内切核酸酶酶切电泳定性其重组效率,qPCR定量分析微环、微质粒及亲本质粒的比例,定量计算重组效率。观察随着诱导时间的推进微环/微质粒值的变化。结果:细菌体内LR克隆酶系统可有效催化亲本质粒的重组,重组率达85%以上。相比商品化LR克隆酶体外反应具有更高的稳定性而且更经济。结论:获得了一种高效、稳定的细菌体内产生位点特异性整合型微环DNA的亲本质粒。Objective： Transgenic technology has an important application value in the field of gene therapy, transgenic animals, genome modification and other biomedical researches. Many researches demonstrated that ФC31 integrase system was an efficient tool for site-specific integration, but the biggest drawback of this system is the integration of the bacterial backbone in the attB donor plasmid. The minicircle DNA is an expression vector containing only the target gene, preparation of minicircle DNA based on ФC31 integrase system is expected to avoid the introduction of bacterial backbone at the integration site, with improved biological safety of the system. The classic procedure for minicircle DNA preparation is laborious with low yield and unstable efficiency. In order to solve above problems, a feasible and effective method for obtaining site-specific integrated minicircle DNA by combining the LR clonase system and the Streptomyces phage ФC31 integrase system were established, and laying a foundation for the construction of transgenic cells without redundant sequences such as bacterial backbone. Methods ： The parental plasmid containing LR clonase system and （I）C31 integrase system is constructed, it can be recombined by L-arabinose induced LR clonase in bacteria to produce a miniplasmid expressing ФC31 integrase in eukaryotic cells and a minicircle DNA containing the original genes such as the target gene and attB site. The recombination efficiency was qualitatively analyzed by restriction endonuclease digestion and quantitativ analyzed by the ratio of calculating the proportion of minicircle DNA, miniplasmid and the parental plasmid. The change ely of minicircle DNA/miniplasmid was observed along with the induction time. Results： A validation plasmid and a parental plasmid for the production of site-specific integrated minicircle DNA were constructed. The LR clonase system could effectively catalyze the recombination of attL and attR in the parental plasmid in bacteria, and the recombination rate wa

关 键 词：微环DNA LR克隆酶系统位点特异性整合 阿拉伯糖启动子ФC31 整合酶 

分 类 号：Q78[生物学—分子生物学]