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  

Construction of Recombinant HCMV with Flag Tagged UL23 Gene via Red-mediated Recombination System

作　　者：胡嘉淼[1] 肖静[1] 刘明亮[1] 曾宝娟[1] 李婧惠[1] 李继东[1] 周天鸿[1] 冉艳红[1] 李弘剑[1] 

机构地区：[1]暨南大学生命科学技术学院生物工程学系,广州510632

出　　处：《生物技术通报》2012年第2期97-101,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(30770106);国家自然科学基金重大研究计划面上项目(90608024);广东省自然科学基金项目(06025162);暨南大学科研培育与创新基金青年基金项目(11611711)

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。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.

关 键 词：UL23人巨细胞病毒 RED同源重组 pKD46 pCP20 

分 类 号：Q78[生物学—分子生物学]