黄瑾,陈相好,吴晓娟,张峥嵘,谷俊莹,陈峥宏,崔古贞
HUANG Jin,CHEN Xianghao,WU Xiaojuan,ZHANG Zhengrong,GU Junying,CHEN Zhenghong,CUI Guzhen

• 1:贵州省普通高等学校病原生物学特色重点实验室
• 2:贵州医科大学基础医学院
• 3:贵州医科大学医学检验学院

摘要(Abstract)：

目的:构建基于细菌Ⅱ型内含子的Targetron基因打靶载体,并在大肠杆菌中验证其功能。方法:以构建的p SY7载体为基础、以大肠杆菌lac Z基因为例,选择lac Z-1683a和lac Z-1874s位点为靶位点,利用在线设计软件设计打靶引物,通过重叠延伸PCR方法获得特异性打靶序列,并将其克隆到p SY7载体,获得p SY7-lacZ-1683a和p SY7-lacZ-1874s打靶载体,转化大肠杆菌后利用异丙基硫代半乳糖苷(IPTG)诱导插入型内含子的表达,最后利用菌落PCR和蓝白斑计数法验证其功能及打靶效率。结果:成功构建了p SY7-lacZ-1683a和p SY7-lac Z-1874s两种Targetron打靶载体,通过0.5 mmol/L IPTG诱导45 min后,菌落PCR显示Ⅱ型内含子能特异性插入到lac Z-1683a、lac Z-1874s位点,蓝白斑计数显示lac Z-1683a位点的打靶效率为(14.299±1.271)%,lac Z-1874s位点的打靶效率为(9.217±1.024)%。结论:构建的Targetron打靶载体能够特异性的插入大肠杆菌染色体靶位点。
Objective:To construct Targetron targeting vector based on bacteria group Ⅱ and verify its function in E.coli.Methods:Based on the constructed pSY7 vector and taking lacZ gene of E.coli as an example,lacZ-1683 a and lac Z-1874 s loci were selected as target sites.Targeting primers were designed by on-line design software.Specific targeting sequences were obtained by overlapping extended PCR and then cloned into p SY7 vectors.The pSY7-lacZ-1683 a and pSY7-lacZ-1874 s targeting vectors were obtained and transformed into E.coli.IPTG was used to induce the expression of insertional group Ⅱ introns.Finally,colony PCR and blue-white spot counting were used to verify the function and targeting efficiency.Results:Two Targetron vectors,pSY7-lac Z-1683 a and pSY7-lacZ-1874 s,were successfully constructed.After 45 minutes of induction by 0.5 mmol/L IPTG,colony PCR showed that Group Ⅱ intron could be specifically inserted into lac Z-1683 a and lacZ-1874 s sites.The targeting efficiency of lacZ-1683 a site was(14.299 ± 1.271) % and that of lac Z-1874 s site was(9.217 ±1.024) %.Conclusion:Targetron vectors constructed in this paper can specifically insert into the target site in E.coli.

关键词(KeyWords)： 型内含子;打靶载体;大肠杆菌;重叠延伸PCR;Targetron基因
group Ⅱ introns;Targetron;targeting vector;Escherichia coli;overlapping extension PCR

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(31760318,31500078,31560318,31601012);; 贵州省科技计划项目[黔科合基础(2018)1132];; 贵州省研究生科研基金立项项目(11348)

作者(Author): 黄瑾,陈相好,吴晓娟,张峥嵘,谷俊莹,陈峥宏,崔古贞
HUANG Jin,CHEN Xianghao,WU Xiaojuan,ZHANG Zhengrong,GU Junying,CHEN Zhenghong,CUI Guzhen

DOI: 10.19367/j.cnki.1000-2707.2019.07.001

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