| 基于Red重组系统构建含Flag标签标记UL23基因的重组人巨细胞病毒 |
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| 引用本文： | 胡嘉淼,肖静,刘明亮,曾宝娟,李婧惠,李继东,周天鸿,冉艳红,李弘剑. 基于Red重组系统构建含Flag标签标记UL23基因的重组人巨细胞病毒[J]. 生物技术通报, 2012, 0(2): 97-101 |
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| 作者姓名： | 胡嘉淼 肖静 刘明亮 曾宝娟 李婧惠 李继东 周天鸿 冉艳红 李弘剑 |
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| 作者单位： | 暨南大学生命科学技术学院生物工程学系,广州,510632 |
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| 基金项目： | 国家自然科学基金,国家自然科学基金重大研究计划面上项目,广东省自然科学基金,暨南大学科研培育与创新基金青年基金 |
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| 摘 要： | 利用来源于λ噬菌体的Red系统,将Flag标签及两侧带有FRT位点的卡那霉素抗性基因片段插入原HCMV TowneBAC中UL23基因3 '末端区域,通过卡那抗性筛选带有抗性标记的重组菌株,并通过表达重组酶FLP的质粒pCP20去除卡那霉素抗性基因,得到带有Flag标签标记UL23基因和单一FRT位点的突变BAC.重组后的BAC分子同质粒pcDNA3.1(+)-pUL82共转染HFF细胞后重建重组HCMV.Western blotting检测证实所构建重组病毒能够表达含Flag标签标记的pUL23蛋白.此含有Flag标签标记UL23基因的重组HCMV的成功构建为了进一步研究人巨细胞病毒UL23基因及其产物的功能提供依据.
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| 关 键 词： | UL23人巨细胞病毒 Red同源重组 pKD46 pCP20 |
Construction of Recombinant HCMV with Flag Tagged UL23Gene via Red-mediated Recombination System |
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| Hu Jiaomiao , Xiao Jing , Liu Mingliang , Zeng Baojuan , Li Jinghui , Li Jidong , Zhou Tianhong , Ran Yanhong , Li Hongjian. Construction of Recombinant HCMV with Flag Tagged UL23Gene via Red-mediated Recombination System[J]. Biotechnology Bulletin, 2012, 0(2): 97-101 |
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| Authors: | Hu Jiaomiao Xiao Jing Liu Mingliang Zeng Baojuan Li Jinghui Li Jidong Zhou Tianhong Ran Yanhong Li Hongjian |
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| Affiliation: | Hu Jiaomiao Xiao Jing Liu Mingliang Zeng Baojuan Li Jinghui Li Jidong Zhou Tianhong Ran Yanhong Li Hongjian(College of Life Science and Technology,Jinan University,Guangzhou 510632) |
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| Abstract: | The DNA fragment of Flag tag and kanamycin-resistant gene flanked with FRT sites was inserted into 3’ end of UL23 gene of HCMV Towne BAC via Red-mediated recombinant system.The positive clones were identified by kanamycin-selection in the first round of recombination and the kanamycin-resistant gene was deleted with the plasmid pCP20 which can express the FLP in the second round.This recombinant BAC was co-transfected with plasmid pcDNA3.1(+)-pUL82 into HFF cell to produce recombinant HCMV.The pUL23 fusion protein was expressed correctly and confirmed by Western blotting,indicating that the recombinant HCMV expressing Flag-tagged-pUL23 has been established,which may provide a powerful tool for the functional research of UL23 gene. |
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| Keywords: | UL23 HCMV Red-mediated recombination pKD46 pCP20 |
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